Method for solution phase synthesis of oligonucleotides and peptides

ABSTRACT

This invention discloses an improved method for the sequential solution phase synthesis of oligonucleotides and peptides. The method lends itself to automation and is ideally suited for large scale manufacture oligonucleotides with high efficiency.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/780,517, filed Jan. 8, 1997, entitled "Method for Solution PhaseSynthesis of Oligonucleotides and Peptides," which is acontinuation-in-part of PCT Application Serial No. PCT/US96/16668, filedon Oct. 17, 1996 designating the United States, entitled "Method forSolution Phase Synthesis of Oligonucleotides."

FIELD OF THE INVENTION

This invention relates to the fields of nucleic acid and peptidechemistry. Specifically, this invention describes a novel method forpreparing oligonucleotides and peptides. The method utilized herein forpreparing said oligonucleotides and peptides is called PASS, an acronymfor Product Anchored Sequential Synthesis.

BACKGROUND OF THE INVENTION

Until quite recently, the consideration of oligonucleotides in anycapacity other than strictly informational was unheard of. Despite thefact that certain oligonucleotides were known to have interestingstructural possibilities (e.g., t-RNAs) and other oligonucleotides werebound specifically by polypeptides in nature, very little attention hadbeen focused on the non-informational capacities of oligonucleotides.For this reason, among others, little consideration had been given tousing oligonucleotides as pharmaceutical compounds.

There are currently at least three areas of exploration that have led toextensive studies regarding the use of oligonucleotides aspharmaceutical compounds. In the most advanced field, antisenseoligonucleotides are used to bind to certain coding regions in anorganism to prevent the expression of proteins or to block various cellfunctions. Additionally, the discovery of RNA species with catalyticfunctions--ribozymes--has led to the study of RNA species that serve toperform intracellular reactions that will achieve desired effects. Andlastly, the discovery of the SELEX process (Systematic Evolution ofLigands by Eponential Enrichment) (Tuerk and Gold (1990) Science249:505) has shown that oligonucleotides can be identified that willbind to almost any biologically interesting target.

SELEX is a method for identifying and producing nucleic acid ligands,termed "nucleic acid antibodies", e.g., nucleic acids that interact withtarget molecules (Tuerk and Gold (1990) Science 249:505). The methodinvolves selection from a mixture of candidate oligonucleotides andstep-wise iterations of binding, partitioning and amplification, usingthe same general selection theme, to achieve virtually any desiredcriterion of binding affinity and selectivity.

The use of antisense oligonucleotides as a means for controlling geneexpression and the potential for using oligonucleotides as possiblepharmaceutical agents has prompted investigations into the introductionof a number of chemical modifications into oligonucleotides to increasetheir therapeutic activity and stability. Such modifications aredesigned to increase cell penetration of the oligonucleotides, tostabilize them from nucleases and other enzymes that degrade orinterfere with the structure or activity of the oligonucleotide analogsin the body, to enhance their binding to targeted RNA, to provide a modeof disruption (terminating event) once sequence-specifically bound totargeted RNA and to improve their pharmacokinetic properties.

Recent research has shown that RNA secondary and tertiary structures canhave important biological functions (Tinoco et al. (1987) Cold SpringHarb. Symp. Quant. Biol. 52:135; Larson et al. (1987) Mol. Cell.Biochem. 74:5; Tuerk et al. (1988) Proc. Natl. Acad. Sci. USA 85:1364;Resnekov et al. (1989) J. Biol. Chem. 264:9953). PCT Patent ApplicationPublication WO 91/14436, entitled "Reagents and Methods for ModulatingGene Expression Through RNA Mimicry," describes oligonucleotides oroligonucleotide analogs which mimic a portion of RNA able to interactwith one or more proteins. The oligonucleotides contain modifiedinternucleoside linkages rendering them nuclease-resistant, haveenhanced ability to penetrate cells, and are capable of binding targetoligonucleotide sequences.

Although there has been a fair amount of activity in the development ofmodified oligonucleotides for use as pharmaceuticals, little attentionhas been paid to the preparation and isolation of these compounds on ascale that allows clinical development. The conventional laboratoryscale 1 μmole automated oligonucleotide synthesis does not provide asufficient amount of the compound of interest to enable clinicaldevelopment. For clinical development oligonucleotides must be producedin gram-scale to multigram scale amounts at a minimum. Although thereare reports of large-scale oligoribonucleotide syntheses in theliterature, the term "large-scale" has been applied to the 1 to 10 μmolescale, rather than gram-scale or kilogram-scale amounts. (Iwai et al.(1990) Tetrahedron 46:6673-6688).

The current state of the art in oligonucleotide synthesis is automatedsolid phase synthesis of oligonucleotides by the phosphoramidite method,which is illustrated in Scheme 1. (Beaucage and Iyer (1992) Tetrahedron48:2223-2311; Zon and Geiser (1991) Anti-Cancer Drug Design 6:539-568;Matteucci and Caruthers (1981) J. Am. Chem. Soc. 103:3185-3191).Briefly, the 3'-terminal nucleoside of the oligonucleotide to besynthesized is attached to a solid support and the oligonucleotide issynthesized by addition of one nucleotide at a time while remainingattached to the support. As depicted in Scheme 1 a nucleoside monomer isprotected (P₁) and the phosphoramidite is prepared (1). Thephosphoramidite (referred to as the 5'-protected monomer unit) is thencovalently attached to the growing oligonucleotide chain (2), via aphosphite triester linkage, through the 5'-hydroxy group of the ribosering of the growing oligonucleotide chain to yield the oligonucleotideproduct (3), in which the majority of the growing oligonucleotide chainhas been extended by one nucleotide, but a significant percent of chainsare not extended. The product (3) is then oxidized to yield thephosphate triester (4). Prior to the addition of the next base to thegrowing nucleotide chain, the 5'-hydroxyl group must be deprotected. Ascan be seen in Scheme 1 (compound 4), however, not all of the reactivesites on the solid support react with the 5'-protected monomer. Theseunreacted sites (referred to as failure sequences) must, therefore, beprotected (referred to as capping) (5) prior to deprotection of the5'-hydroxyl group (6). Subsequent monomers, which have also beenprotected and converted to the phosphoramidite, are then sequentiallyadded by coupling the 5'-end of the growing oligomer to the 3'-end ofthe monomer. Each coupling reaction extends the oligonucleotide by onemonomer via a phosphite triester linkage. At each step--and in the caseof the initial reaction with the solid support--there are reactive sitesthat fail to react with the 5'-protected monomer, which results inoligonucleotides that have not been extended by one nucleotide monomer(failure sequences). When the synthesis is complete the desiredoligonucleotide (6(n+1 sequence)) is deprotected and cleaved from theresin, together with all of the failure sequences (n, n-x).

The yield of conventional solid phase oligonucleotide synthesisdecreases exponentially with the number of monomers coupled. Thisincreases the difficulty of purifying the crude product away from thefailure sequences. Additionally, even after high resolution purificationhas been achieved, it remains very difficult to verify the sequence andcomposition of the product, especially if it contains non-standardnucleotides. ##STR1##

Automated oligonucleotide synthesis on solid supports is very efficientfor the preparation of small amounts, 0.001 to 0.01 mmol, of a varietyof sequences in a minimum amount of time with reasonable yield. It is,however, a highly inefficient process in terms of overall process yieldbased on input monomer. Typically a 16 fold excess of phosphoramidite isnecessary per monomer addition. It has been recognized that theautomated solid phase synthesis approach does not readily lend itself tobe scaled to a level that allows efficient manufacture ofoligonucleotide pharmaceuticals. (Zon and Geiser (1991) Anti-Cancer DrugDesign 6:539-568).

The inefficiency of the solid phase synthesis is created to a largeextent by the heterophase monomer coupling reaction and by the covalentattachment of both unreacted failure sequences and reaction product tothe same support bead. In each cycle, 1-5% of the nucleotide bound tothe support does not react with the activated monomer. These unreactedcompounds, referred to as failure sequences, as discussed above, must beblocked or capped in order to prevent the subsequent addition ofmonomers to incomplete oligonucleotides. The generation of failuresequences at every step of the synthesis produces a crude productcontaminated with highly homologous byproducts, which must be carriedthrough to the final crude product (see Scheme 1, structure 6 (n, n-x)).As a result, purification of crude synthetic oligonucleotides to a stateacceptable for clinical studies is extremely cumbersome and inefficient.To minimize the percent of failure sequences, a large excess of monomer(approximately 16 fold) is used.

A method to scale-up solid phase oligonucleotide synthesis using ahigher loaded polystyrene support was reported by Montserrat et al.(1994) Tetrahedron 50:2617-2622. This method, however, does not overcomethe primary problem associated with solid phase synthesis, in that aconsiderable monomer excess is still required to minimize failuresequences. Additionally, the method does not provide consistentlysatisfactory yields.

In an attempt to decrease the excess of monomer needed to achievecoupling and to achieve easy scaleability, Bonora et al. (1993) NucleicAcids Res. 21:1213-1217, have investigated using polyethylene glycol(PEG) as a 3'-support that is soluble in the monomer coupling reaction.This method has been used to prepare oligonucleotides by phosphoramiditecoupling, H-phosphonate condensation and phosphotriester condensation.(See Bonora (1987) Gazzetta Chimica Italiana 117:379; Bonora et al(1990) Nucleic Acids Res. 18:3155; Bonora et al. (1991) Nucleosides &Nucleotides 10:269; Colonna et al. (1991) Tetrahedron Lett.32:3251-3254; Bonora and Scremin (1992) Innovation Perspect. Solid PhaseSynth. Collect. Pap., Int. Symp., 2nd, "Large Scale Synthesis ofOligonucleotides. The HELP Method: Results and Perspectives", pp.355-358, published by Intercept, Andover, UK; Scremin and Bonora (1993)Tetrahedron Lett. 34:4663; Bonora (1995) Applied Biochemistry andBiotechnology 54:3; Zaramella and Bonora (1995) Nucleosides &Nucleotides 14:809). The weakness of this approach is the unacceptablylow recovery of support bound oligonucleotide after each reaction step.Additionally, this method does not address the problem of failuresequences that must be capped and carried through to the final product.

A polyethylene glycol-polystyrene copolymer support has also been usedfor the scale-up of oligonucleotide synthesis. (Wright et al. (1993)Tetrahedron Lett. 34:3373-3376). At the 1 mmol scale a 96.6% couplingefficiency per monomer addition was reported for an 18mer DNA. Again,this method does not address the problem of failure sequences bound tothe resin.

Zon et al. have suggested a block approach to the synthesis ofoligonucleotides, in which a library of dimer or multimeroligonucleotide fragments are prepared in solution and then coupled toeach other. (Zon and Geiser (1991) Anti-Cancer Drug Design 6:539-568).The fragments are activated for coupling by differential 5'-deprotectionand 3'-phosphorylation. Phosphotriester coupling has been suggested forfragment preparation. (Bonora et al. (1993) Nucleic Acids Res.21:1213-1217). Due to the comparatively low yield of phosphotriestercoupling this approach has not been widely accepted.

In conventional oligonucleotide synthesis, the 5'-protecting groupserves to prevent reaction of the 5'-hydroxyl group of one monomer withthe phosphoramidite group of a second monomer during the coupling step.The 4,4'-dimethoxytrityl (DMT) group is commonly used as the5'-protecting group (Schaller et al. (1963) J. Am. Chem. Soc. 85:3821)of the 5'-protected monomer unit added during oligonucleotide synthesis.This group is chosen because of the ease and selectivity with which itcan be removed from the 5'-oxygen of the oligonucleotide product priorto addition of the next 5'-protected monomer unit (for a review see:Beaucage and Iyer (1992) Tetrahedron 48:2223-2311). In solution,deprotection of the 5'-DMT group is impaired by the reversibility ofacid induced detritylation. In order to drive this reaction tocompletion, a scavenger of the free trityl cation is added forsolution-phase detritylation (Ravikumar et al. (1995) Tetrahedron Lett.36:6587). It has been recognized that the final 5'-terminal DMT groupmay serve as a hydrophobic handle which allows separation of thefull-length product oligonucleotide from shorter failure sequences byreverse phase chromatography. Additionally, highly hydrophobic analogsof the DMT group have been prepared to enhance the resolution of theseparation of full length deprotected oligonucleotide product fromfailure sequences after complete solid phase synthesis (Seliger andSchmidt (1987) Journal of Chromatography 397:141). In another approach,a fluorescent trityl analog has been used for the 5'-terminal protectinggroup during oligonucleotide synthesis to allow facile detection offull-length product in crude deprotected oligonucleotide (Fourrey et al.(1987) Tetrahedron Lett. 28:5157). Colored trityl groups were devised toallow monitoring of specific monomer additions during solid phaseoligonucleotide synthesis (Fisher and Caruthers (1983) Nucleic AcidsRes. 11:1589). Other modified trityl groups have been prepared for thepurpose of changing or enhancing the selectivity with which the tritylgroup can be removed from the oligonucleotide during solid phaseoligonucleotide synthesis (for a review, see: Beaucage and Iyer (1992)Tetrahedron 48:2223-2311).

To date, trityl groups which allow anchoring of the product to a resinor membrane during oligonucleotide synthesis in solution have not beendesigned. Additionally, trityl groups which can covalently react with aderivatized resin, membrane or soluble polymer have not been reported.

Proteins and peptides play a critical role in virtually all biologicalprocesses, functioning as enzymes, hormones, antibodies, growth factors,ion carriers, antibiotics, toxins, and neuropeptides. Biologicallyactive proteins and peptides, therefore, have been a major target forchemical synthesis. Chemical synthesis is used to verify structure andto study the relationship between structure and function, with the goalof designing novel compounds for potential therapeutic use. Syntheticpeptides comprise a prominent class of pharmaceuticals.

There are currently two basic methods for synthesizing proteins andpeptides: solution phase synthesis in which the chemistry is carried outin solution and solid phase synthesis in which the chemistry is carriedout on a solid support. A major disadvantage of solution phase synthesisof peptides is the poor solubility of the protected peptideintermediates in organic solvents. Additionally, purifications aredifficult and time consuming. Solid phase synthesis overcomes theseproblems and has become the method of choice in synthesizing peptidesand proteins. (See, Merrifield (1963) J. Am. Chem. Soc. 8:2149; Mitchellet al. (1978) J. Org. Chem. 43:2845-2852; Bodansky (1984) in Principlesof Peptide Synthesis, (Springer Verlag Berlin); Stewart and Young (1984)in Solid Phase Peptide Synthesis, sec. ed., Pierce Chemical Company,Illinois pp. 88-95).

Generally, solid phase peptide synthesis proceeds from the C-terminal tothe N-terminal amino acid. Briefly, the carboxy-terminal amino acid ofthe peptide to be synthesized is protected and covalently attached to asolid support, typically a resin. The subsequent amino acids (which havebeen N-protected and side-chain protected) are then sequentially addedeither as the free carboxylic acid or in the form of an activated esterderivative. The two most frequently used protecting groups for theN-terminal amino group are Fmoc (Fmoc-9-fluorenylmethyl carbonyl;Carpino and Han (1972) J. Org. Chem. 37:3404) and Boc(Boc-tert-butoxycarbonyl; Sheppard (1986) Science 33:9; Pulley andHegedus (1993) J. Am. Chem. Soc. 115:9037-9047). When the synthesis iscomplete the peptide is deprotected, cleaved from the resin andpurified.

Increasing the efficiency of solution phase preparation of peptidescontinues to be an active field of investigation. Introduction of theN-Fmoc protected amino acid fluorides and subsequent in situ generationof these monomers allows efficient solution phase preparation ofpeptides with minimal racemization. (Carpino et al. (1990) J. Am. Chem.Soc. 112:9651-9652; Carpino and El-Faham (1995) J. Am. Chem. Soc.117:5401-5402). This method has been applied to the preparation of theantibiotic vancomycin carboxamide derivatives. (Sundram and Griffin(1995) J. Org. Chem. 60:1102-1103).

Although there have been continuous improvements in the methods forpeptide synthesis, typical yields for synthetic peptides remain rathermoderate. This necessitates lengthy downstream processing procedures toobtain pure product.

The Diels-Alder reaction is a cycloaddition reaction between aconjugated diene and an unsaturated molecule to form a cyclic compoundwith the π-electrons being used to form the new σ bonds. The Diels-Alderreaction is an example of a [4+2] cycloaddition reaction, as it involvesa system of 4π electrons (the diene) and a system of 2-π electrons (thedienophile). The reaction can be made to occur very rapidly, under mildconditions, and for a wide variety of reactants. The Diels-Alderreaction is broad in scope and is well known to those knowledgeable inthe art. A review of the Diels-Alder reaction can be found in "AdvancedOrganic Chemistry" (March, J., ed.) 761-798 (1977) McGraw Hill, NY,which is incorporated herein by reference.

To date, although a number of attempts have been made, there stillremains a need for a method to produce oligonucleotides in largequantities, in continuous operations, at low cost and without laboriouspurification.

BRIEF SUMMARY OF THE INVENTION

The present invention includes methods for the sequential solution phasesynthesis of oligonucleotides that increase reaction yields and allowfor scale-up possibilities. As opposed to traditional schemes in whichthe 3'-end of the growing oligonucleotide is bound to a solid support,the present invention is characterized by use of an anchor groupattached to the 5'-end of the growing oligonucleotide that allowssuccessfully coupled product to be separated from unreacted startingmaterials. In one embodiment, the anchor group also serves as the 5'--OHprotecting group and the coupling reaction occurs in solution. Thesuccessfully reacted oligomer will contain the protecting group, whilethe unreacted oligomer will not, and the materials can be partitionedbased on the presence of the anchor/protecting group. In a preferredembodiment, the anchor group reacts covalently with a derivatized solidsupport, such as a resin, membrane or polymer.

In a preferred embodiment of the invention, the monomer unit consists ofa 5'-protected phosphoramidite or H-phosphonate, wherein the protectinggroup is a substituted trityl group, levulinic acid group, or silylether group. In one embodiment, the unreacted oligonucleotide startingmaterial (failure sequence) may be separated from the reactedoligonucleotide product based on the affinity of the protecting groupfor a chromatography resin. In a preferred embodiment, the unreactedoligonucleotide starting material may be separated from the reactedoligonucleotide product based on the specific reaction of the protectinggroup with a derivatized solid support, such as a resin, membrane orpolymer. In a preferred aspect of the invention the partitioning methodto remove unreacted oligonucleotide serves to allow for isolation andreuse of the unreacted oligonucleotide and also will allow the reactedoligonucleotide to be deprotected in preparation for the subsequentaddition of the next 5'-protected monomer unit.

The method of this invention is not limited to phosphoramidite couplingchemistry, but is compatible with other coupling reactions such asH-phosphonate or phosphate triester coupling chemistry. This method alsolends itself to automation and is ideally suited for the large scalemanufacture of oligonucleotides with high efficiency.

The present invention includes a method and apparatus to automaticallyseparate the product from the unreacted 5'-protected monomer unit andthe starting material. In one embodiment the apparatus is comprised ofan extraction vessel and a chromatography resin filtration chamber,which contains a solid support. Upon completion of a monomer additionreaction, the reaction mixture is pumped into the extraction chamber,extracted and then eluted through the solid support, which retains onlythe 5'-protected monomer unit. The product is then separated from thestarting material by eluting through a solid support that retains onlythe product. In a second embodiment the chromatography resin filtrationchamber contains a solid support which covalently reacts with both the5'-protected monomer unit and the product. The starting material iseluted from the solid support and the monomer and product are thenreleased from the solid support with a dilute acid. The product is thenseparated from the 5'-protected monomer unit by passage through anultrafiltration membrane.

A material cost analysis reveals that the 5'-protected phosphoramiditeis the most costly reaction component in oligonucleotide synthesis. Thecost of the remaining materials are trivial in comparison. Therefore, itwould be desirable to make the monomer the limiting reagent.Furthermore, a particular intermediate oligonucleotide sequence whichfailed to add to the incoming monomer could serve as an intermediate ina subsequent synthesis. Using the method of this invention, verificationof the sequence and composition of oligonucleotide product becomestrivial. After every monomer addition cycle, a fully protected, neutralintermediate is obtained, which is easily analyzed by mass spectrometrywithout tedious sample preparation. Over the course of anoligonucleotide synthesis a library of analytical data for everysequential monomer addition can be obtained. Thus, product analysisbecomes an integral part of the process.

The present invention also includes methods for the sequential solutionphase synthesis of peptides. This embodiment of the invention ischaracterized by use of an anchor group attached to the N-terminalprotecting group of the growing peptide that allows successfully coupledproduct to be separated from unreacted starting materials. Thesuccessfully reacted peptide will contain the anchor group, while theunreacted peptide will not, and the materials can be partitioned basedon the presence of the anchor/protecting group. In a preferredembodiment, the anchor group reacts covalently with a derivatized solidsupport, such as a resin, membrane or polymer. The invention provides amethod for the solution phase synthesis of a wide variety of peptidesand modified peptides.

The methods of the present invention can be extended to all sequentialpolymerization reactions and thus to the sequential synthesis of anypolymer, including but not limited to peptide nucleic acids (PNAs) andcarbohydrates.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the reverse phase High Pressure Liquid Chromatography(HPLC) trace of the phosphoramidite coupling reaction mixture set forthin Example 1 prior to oxidation.

FIG. 2 illustrates the reverse phase HPLC trace of the phosphoramiditecoupling reaction set forth in Example 1 after oxidation. The postoxidation trace has been superimposed on the preoxidation trace of FIG.1.

FIG. 3 illustrates the reverse phase HPLC traces of a mixture ofoxidized phosphoramidite coupling reaction set forth in Example 1, bothprior to and after being passed through a DEAE Sephadex® filter plug.

FIG. 4 illustrates the reverse phase HPLC traces of the oxidizedphosphoramidite coupling reaction set forth in Example 1, after beingeluted through a C18 resin with water/acetonitrile and after treatmentwith acetic acid and elution with water/acetonitrile.

FIG. 5 illustrates schematically an automated extraction and filtrationsystem designed for use with the method of this invention.

FIG. 6 illustrates the anion exchange HPLC trace of the 15 baseoligonucleotide prepared in Example 7 using 3'-PEG anchored solutionphase synthesis.

FIG. 7 illustrates graphically the Diels-Alder capture data for thereaction of 5'-DHDTO-T-[3',3']-T-OSiPDBT-5' with polystyrene maleimideresins containing 1.0 eq, 2.5 eq, 5 eq and 10 eq of maleimide.

FIG. 8 illustrates schematically an automated extraction and filtrationsystem designed for use with Diels-Alder product capture.

FIG. 9 illustrates graphically the precipitation and centrifugation ofPEG-precipitated by ethyl ether, isopropyl ether and N-butyl ether.

FIG. 10 illustrates the assembly of a peptide using Product AnchoredSequential Synthesis (PASS).

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes methods for the solution phase synthesisof oligonucleotides, referred to herein as Product Anchored SequentialSynthesis (PASS). Unlike traditional schemes where the 3'-end of thegrowing oligonucleotide is bound to a solid support, the presentinvention is characterized by utilization of an anchor group attached tothe 5'-end of the growing oligonucleotide product that allowssuccessfully coupled product to be separated from unreacted startingmaterials. In a preferred embodiment the anchor group also serves as the5'-OH protecting group. The successfully reacted oligonucleotide productwill contain the protecting group, while the unreacted oligonucleotidestarting material will not, and the product can be partitioned away fromthe starting material based on the presence of the blocking/protectinggroup. Unreacted starting material is recovered and can be reused in asubsequent synthesis batch of the same oligonucleotide. Thus, incontrast to conventional solid phase synthesis, the improved method foroligonucleotide synthesis described herein does not employ a solidsupport for anchoring of the 3'-end of the growing oligonucleotidechain.

Specifically, the invention provides a method for the solution phasesynthesis of a wide variety of oligonucleotides and modifiedoligonucleotides comprising reaction of a 5'-protected monomer unit withthe 5'-end of a growing oligonucleotide chain in solution. Performingthese reactions in solution, rather than on solid supports, provides forbetter reaction kinetics. In an additional aspect of the invention,following reaction between the 5'-protected monomer unit and the growingoligonucleotide, the unreacted 5'-protected monomer unit may beactivated and oxidized to form a charged species that may be easilypartitioned from the remainder of the reaction medium. In the preferredembodiments of the invention the monomer units are phosphoramidites,that upon oxidation can be easily converted to phosphate diesters. Thecharged phosphate species can be easily partitioned from the remainderof the reaction medium. Additionally, in a preferred embodiment theoxidation may be performed in situ.

When using a H-phosphonate as the 5'-protected monomer unit, which is acharged species (Example 2, Scheme 5), the oxidization of H-phosphonateis deferred until after the addition of the final monomer. The chargedH-phosphonate monomers, produce neutral H-phosphonate diester productsafter coupling, and the charged monomer species are readily removed byanion exchange filtration or extraction. In addition, the recoveredH-phosphonate monomers are reusable.

The 5'-protecting group that is utilized can be selected from any classof chemical functionalities that meets the basic requirements of theinvention. The protecting group must be of a type that can be used todifferentiate the product of the reaction from the remainder of thereaction mixture in order to effect a separation. Preferably, theprotecting group will have a strong affinity for or a reactivity with aparticular phase or solid support and it must be easily cleaved orremoved from the phase or solid support with high selectivity. Theoligonucleotide product may be separated from unreacted oligonucleotidestarting material using standard methods known to those skilled in theart including, but not limited to, centrifugation, separation on aresin, silica gel based separation, separation based on affinity for ametal, separation based on a magnetic force or electromagnetic force orseparation based upon covalent attachment to a suitable solid support.

In a preferred aspect of the invention the partitioning method to removeunreacted oligonucleotide starting material serves to both allow for theisolation for reuse of the unreacted oligonucleotide and also willresult in a resin-bound oligonucleotide product which is easilydeprotected in preparation for the subsequent addition of the next5'-protected monomer unit. Most preferably, the protecting group willcovalently react with a derivatized solid support, such as a resin,membrane or polymer, to give a covalently anchored protecting groupwhich may easily be cleaved from the oligonucleotide with highselectivity.

In the most preferred embodiment of the invention, the monomer unitconsists of a 5'-protected phosphoramidite or H-phosphonate, wherein theprotecting group is a substituted trityl group, levulinic acid group orsilyl ether group. The preferred substitution on the protecting group isa diene functionality, which can react, via a Diels-Alder reaction, witha solid support, such as a resin, membrane or polymer that has beenderivatized with a dienophile. In this embodiment, the unreactedoligonucleotide starting material is separated from the reactednucleotide product based on the selective or specific covalent reactionof the 5'-protecting group with a derivatized resin.

The present invention also includes methods for the solution phasesynthesis of peptides by PASS. This method is characterized byutilization of an anchor group attached to the N-terminal amino acid endof the growing peptide product that allows successfully coupled productto be separated from unreacted starting materials. In a preferredembodiment the anchor group also serves as the N-protecting group. Thesuccessfully reacted peptide product will contain the anchor group,while the unreacted peptide starting material will not, and the productcan be partitioned away from the starting material based on the presenceof the anchor group. Unreacted starting material is recovered and can bereused in a subsequent synthesis batch of the same peptide. Thus, incontrast to conventional solid phase synthesis, the improved method forpeptide synthesis described herein does not employ a solid support foranchoring of the carboxy-terminal end of the growing peptide chain.

Specifically, the invention provides a method for the solution phasesynthesis of a wide variety of peptides and modified peptides comprisingreaction of an N-protected amino acid monomer unit with the N-terminalend of a growing peptide chain in solution. The method for peptidesynthesis described herein is designed to introduce highly efficient andscalable preparation of peptides with unprecedented purity. This isachieved by exploiting the N-terminal monomer protecting group tofunction as a handle which allows selective and efficient isolation ofthe peptide product at each amino acid addition step (Product AnchoredSequential Synthesis, PASS).

The N-terminal protecting group that is utilized can be selected fromany class of chemical functionalities that meets the basic requirementsof the invention. The protecting group must be of a type that can beused to differentiate the product of the reaction from the remainder ofthe reaction mixture in order to effect a separation. Preferably, theprotecting group will have a strong affinity for or a reactivity with aparticular phase or solid support and it must be easily cleaved orremoved from the phase or solid support with high selectivity. Theprotecting group must also be compatible with conventional peptidesynthesis steps. The peptide product may be separated from unreactedpeptide starting material using standard methods known to those skilledin the art including, but not limited to, centrifugation, separation ona resin, silica gel based separation, separation based on affinity for ametal, separation based on a magnetic force or electromagnetic force orseparation based upon covalent attachment to a suitable solid support.

In a preferred aspect of the invention the partitioning method to removeunreacted peptide starting material serves to both allow for theisolation for reuse of the unreacted peptide and also will result in aresin-bound peptide product which is easily deprotected in preparationfor the subsequent addition of the next N-terminal protected amino acidmonomer unit. Most preferably, the protecting group will covalentlyreact with a derivatized solid support, such as a resin, membrane orpolymer, to give a covalently anchored protecting group which may easilybe cleaved from the peptide with high selectivity.

Certain terms used to describe the invention herein are defined asfollows:

"Nucleoside" means either a deoxyribonucleoside or a ribonucleoside orany chemical modifications thereof. Modifications of the nucleosidesinclude, but are not limited to, 2'-position sugar modifications,5-position pyrimidine modifications, 8-position purine modifications,modifications at cytosine exocyclic amines, substitution of5-bromo-uracil, and the like.

"Oligonucleotide" refers to either DNA or RNA or any chemicalmodifications thereof. The oligonucleotides synthesized by the method ofthis invention are depicted generally as follows: ##STR2## wheren=1-1,000, A is a 2'-sugar substituent as defined below and B is anucleobase as defined below.

A "solid support" as used herein refers to a resin, membrane, phase,polymer, polymer precursor, or soluble polymer that can undergo phasetransition. A solid support also refers to a resin, membrane, phase,polymer, polymer precursor, or soluble polymer that has been derivatizedwith a D group or a Y group, as defined below. The term resin and solidsupport are used interchangeably and one of ordinary skill in the artwill recognize what is intended by the term resin. Examples of solidsupports include, but are not limited to, maleimide derivatizedpolystyrene, polystyrene derivatized with D or Y groups, as definedbelow, dienophile or diene derivatized polystyrene, Tentagel™derivatized with a D or Y groups, as defined below, dienophile or dienederivatized Tentagel™, dienophile or diene derivatized ultrafiltrationmembranes, dienophile or diene derivatized polyethylene glycol, diene ordienophile derivatized inorganic oxides, such as silica gel, alumina,controlled pore glass and zeolites, other dienophile or dienederivatized polymers, hydrophobic reverse phase resins, such as C2 toC18 polystyrene, thiopropyl Sepharose (Pharmacia Biotech), mercuratedresin, agarose adipic acid hydrazide (Pharmacia Biotech), or avidinresin.

A "dienophile" is defined as a molecule bearing an alkene group, or adouble bond between a carbon and a heteroatom, or a double bond betweentwo heteroatoms, which can undergo a [2+4] cycloaddition reaction with asuitable diene.

A "diene" is defined as a molecule bearing two adjacent double bonds,where the atoms forming these double bonds can be carbon or aheteroatom, which can undergo a [2+4] cycloaddition reaction with adienophile.

The dienophile can be any group, including but not limited to, asubstituted or unsubstituted alkene, or a substituted or unsubstitutedalkyne. Typically, the dienophile is a substituted alkene of the formulaC═C--Z or Z'--C═C--Z, wherein Z and Z' are electron withdrawing groupsindependently selected from CHO, COR, COOH, COCl, COAr, CN, NO₂, Ar, CH₂OH, CH₂ Cl, CH₂ NH₂, CH₂ CN, CH₂ COOH, halogen, or C═C.

A "dienophile derivatized solid support" refers to a solid support thathas been functionalized with a dienophile and a "diene derivatized solidsupport" refers to a solid support that has been functionalized with adiene. Preferred solid supports are inorganic oxides selected from thegroup consisting of silica, alumina, zeolites, controlled pore glass,that have hydroxyl groups that are capable of being functionalized, ororganic supports such as polystyrene, as illustrated in Schemes 13 and14. In a preferred embodiment the dienophile is maleimide and the dieneis 3,5-hexadiene.

The "5'-protected monomer units" of this invention are generallydepicted as follows including the conventional numbering for the ribosering: ##STR3## B is a nucleobase; A and A' are 2'-sugar substituents;

W is independently selected from the group consisting of aphosphoramidite, H-phosphonate, phosphotriester, phosphoramidate,protected oligonucleotide and methyl-phosphonate; and

D-E is an alcohol protecting group(s) which serves as an anchor forpartitioning the successfully reacted oligonucleotide product away fromthe unreacted oligonucleotide starting material.

Other obvious substitutions for the substituents described above arealso included within the scope of this invention, which is not limitedto the specific, but rather the generalized formula of reaction.

In a preferred embodiment of the invention:

W is a phosphoramidite or H-phosphonate;

A and A' are independently selected from the group consisting of H, ² H,³ H, Cl, F,

OH, NHOR¹, NHOR³, NHNHR³, NHR³,═NH, CHCN, CHCl₂, SH, SR³, CFH₂, CF₂ H,CR² ₂ Br, --(OCH₂ CH₂)_(n) OCH₃, OR⁴ and imidazole (see U.S. patentapplication Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "NovelMethod of Preparation of 2'Modified Pyrimidines IntramolecularNucleophilic Displacement," which is incorporated herein by reference);

R¹ is selected from the group consisting of H and an alcohol protectinggroup;

R² is selected from the group consisting of ═O, ═S, H, OH, CCl₃, CF₃,halide, optionally substituted C₁ -C₂₀ alkyl (including cyclic, straightchain, and branched), alkenyl, aryl, C₁ -C₂₀ acyl, benzoyl, OR⁴ andesters;

R³ is selected from the group consisting of R², R⁴, CN, C(O)NH₂,C(S)NH₂, C(O)CF₃,

SO₂ R⁴, amino acid, peptide and mixtures thereof;

R⁴ is selected from the group consisting of an optionally substitutedhydrocarbon (C₁ -C₂₀ alkyl, C₂ -C₂₀ alkenyl, C₂ -C₂₀ alkynyl), anoptionally substituted heterocycle, t-butyldimethylsilyl ether,triisopropylsilyl ether, nucleoside, carbohydrate, fluorescent label andphosphate; most preferably A is selected from the group consisting of H,OH, NH,, Cl, F, NHOR³, OR⁴, OSiR⁴ ₃. (See U.S. patent application Ser.No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method ofPreparation of 2'Modified Pyrimidines Intramolecular NucleophilicDisplacement," filed Jun. 22, 1994);

D-E can be any group that enables the partitioning of the "growingoligonucleotide chain" or "oligonucleotide product" away from unwantedside products and starting materials. The partitioning can be done byany suitable method, including but not limited to, silica gel basedchromatography, centrifugation, or any other means known by those in theart for partitioning materials. The preferred method for partitioning isby binding to a resin. The most preferred method for partitioning is bycovalent reaction between D and a derivatized solid support, such as aderivatized resin, polymer, or membrane. The protecting group D-E,therefore, is preferably designed such that D has a strong affinity fora particular resin or phase, and E is designed such that the 5'-oxygen-Ebond is easily cleaved with high selectivity. In cases where E showshigh affinity for a resin or phase, D may be omitted. Most preferablythe protecting group D-E is designed such that D can selectively orspecifically form a covalent bond to a particular derivatized resin,polymer, or membrane.

E includes, but is not limited to, the trityl group or the levulinicacid group or a silyl ether group, as depicted below. ##STR4##

D includes, but is not limited to, groups independently selected from H,OR⁴, an alkyl or substituted alkyl group bearing a conjugated dieneunit, an alkoxy or substituted alkoxy group bearing a conjugated dieneunit, CH₂ ═CHCH═CHCH₂ CH₂ O--, maleimide substituted alkoxy groups,dienophile substituted alkoxy groups, alkoxy groups, an alkylamino orsubstituted alkylamino group bearing a conjugated diene unit, maleimidesubstituted alkylamino groups or substituted alkylamino groups, analkylamino group or substituted alkylamino group bearing a dienophilemoiety, disulfides, aldehydes, and metal chelators, some examples ofwhich are depicted below. The alkyl groups on the above listedsubstituents can have between 1-50 carbons, preferably 1-30 carbons.##STR5##

For the purposes of this invention "nucleobase" will have the followingdefinition. A nucleobase is a purine or a pyrimidine base. Nucleobaseincludes all purines and pyrimidines currently known to those skilled inthe art or any chemical modifications thereof. The purines are attachedto the ribose ring through the nitrogen in the 9 position of the purinering and the pyrimidines are attached to the ribose ring through thenitrogen in the 1 position of the pyrimidine ring. The pyrimidine can bemodified at the 5- or 6- position of the pyrimidine ring and the purinecan be modified at positions 2-, 6- or 8- of the purine ring. Certainmodifications are described in copending U.S. patent applications Ser.Nos. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method ofPreparation of Known and Novel 2'Modified Pyrimidines IntramolecularNucleophilic Displacement" and 08/458,421, filed Jun. 2, 1994, entitled"Palladium Catalyzed Nucleoside Modifications--Methods UsingNucleophiles and Carbon Monoxide" and U.S. Pat. No. 5,428,149, entitled"Method for Palladium Catalyzed Carbon-Carbon Coupling and Productions"which are herein incorporated by reference. More specifically anucleobase includes, but is not limited to, uracil, cytosine,N4-protected cytosine, 4-thiouracil, isocytosine, 5-methyluracil(thymine), 5-substituted uracils, adenine, N6-protected adenine,guanine, N2-protected guanine 2,6-diaminopurine, halogenated purines aswell as heterocycles meant to mimic the purine or pyrimidine ring, suchas imidazole.

The term "peptide" as used herein refers to a polymer of amino acidschemically bound by amide linkages (CONH). An "amino acid" is defined asan organic molecule containing both an amino group (NH₂) and acarboxylic acid (COOH). Specifically an "amino acid" is any compound ofthe general formula R⁵ CH(NH₂)COOH (α-amino acid), wherein R⁵ isselected from the group consisting of H or any suitably protected knownamino acid side chain. Suitable protection for amino acid side chains isknown to those skilled in the art. As used herein the term "peptide"includes peptides, polypeptides and proteins. The peptides synthesizedby the method of this invention are depicted generally as follows:##STR6## wherein n=1-500, and R⁵ is as defined above.

The "N-protected amino acid monomer units" of this invention aregenerally depicted as follows: ##STR7## wherein R⁵ is as defined above;and A-X is a nitrogen protecting group(s) which serves as an anchor forpartitioning the successfully reacted peptide product away from theunreacted peptide starting material. A-X can be any group that enablesthe partitioning of the "growing peptide chain" or "peptide product"away from unwanted side products and starting materials and iscompatible with conventional peptide synthesis. In a preferredembodiment A is selected from the many reported N-protecting groupswhich are known to those in the art, including but not limited tourethanes, such as Fmoc and Boc, benzyl groups, acyl groups, ortriphenylmethyl groups. X is designed to react with a substituent Y on aresin with high selectivity. X is selected from groups such as dienes,in particular 3,5-hexadienoxy or sorbic amide, dienophiles, inparticular maleimide, alkynes, silylether protected diols anddisulfides. The corresponding substituent Y is chosen to be a selectivecovalent reaction partner for substituent X, such as a dienophile,diene, mercaptane, or borate.

"Starting material" as used herein refers to the compound that isreacted with the 5'-protected monomer unit during each cycle of PASS toproduce an oligomer that has been extended by one or more nucleotides.The starting material can be designed to produce a [5',3'] linkagebetween nucleotides or a [3',3'] linkage between nucleotides, dependingon the desired oligonucleotide product. In the first instance thestarting material is a 5'-deprotected otherwise protectedoligonucleotide of length n, in the second case the starting material isa 3'-deprotected otherwise protected oligonucleotide of length n.Typically the starting material is a 5'-deprotected otherwise protectedoligonucleotide of length n, wherein n is an integer from 1-1000. Thestarting material is 2',3'-protected by protecting groups, such as baselabile groups, that are compatible with the reaction of the 5'-protectedmonomer units with the starting material and with 5'-deprotectionreactions. Additionally, because the PASS process consists of thecontrolled and sequential polymerization of an oligonucleotide, thestarting material of one PASS cycle is typically the deprotected productfrom the previous PASS cycle. Because the PASS process does not requirethat the 3'-terminal nucleotide be anchored to a solid support, thestarting material can include non-nucleoside modifications.Non-nucleoside modifications can be introduced to the 3'-terminus whichwould not ordinarily be possible by solid phase synthesis.Non-nucleoside modifications to the 3'-terminus of the starting materialinclude, but are not limited to, the use of polyethylene glycolmono-methylether (molecular weight 5,000 to 100,000) (PEG) or other highmolecular weight non-imnmunogenic units as the 3'-terminal monomer forpreparation of oligonucleotides with improved pharmacokineticproperties.

"Peptide starting material" refers to the compound that is reacted withN-protected amino acid monomer unit during each cycle of PASS to producea peptide product that has been extended by one or more amino acids. Inthis instance starting material is an N-terminal deprotected otherwiseprotected peptide of length n, wherein n is an integer from 1-500.Additionally, because the PASS process consists of the controlled andsequential polymerization of a peptide, the starting material of onePASS cycle is typically the deprotected product from the previous PASScycle. The peptide is protected using protecting groups and methodsknown to those reasonably skilled in the art. The carboxy terminalprotecting group can be selected from a standard protecting group, asoluble polymer or a diagnostic detector.

"Product" as used herein refers to an oligonucleotide that is producedby the covalent reaction of the 5'-protected monomer unit with thestarting material during each PASS cycle. As stated above, if thestarting material is a 5'-deprotected oligonucleotide of length n andthe 5'-monomer unit is a single nucleotide, the product of the reactionwill be a 5'-protected oligonucleotide of length n+1. If the5'-protected monomer unit is an oligonucleotide block of length m theproduct of the reaction will be a 5-protected oligonucleotide of lengthn+m. The product from a particular PASS cycle is then 5'-deprotected andbecomes the starting material for the next cycle.

"Peptide Product" as used herein refers to a peptide that is produced bythe covalent reaction of the N-protected amino acid monomer unit withthe peptide starting material during each PASS cycle. If the peptidestarting material is a N-terminal deprotected peptide of length n andthe N-protected amino acid monomer unit is a single amino acid, theproduct of the reaction will be an N-terminal protected peptide oflength n+1. If the N-protected amino acid monomer unit is a peptideblock of length m the product of the reaction will be an N-terminalprotected peptide of length n+m. The product from a particular PASScycle is then N-deprotected and becomes the starting material for thenext cycle.

A "failure sequence" refers to the starting material from a particularPASS cycle that fails to react with the 5'-protected monomer unit orN-protected amino acid monomer unit during that cycle.

"Growing oligonucleotide chain" refers to either a 5'-deprotectedoligonucleotide chain or a 5'-protected oligonucleotide chain that hasbeen prepared by the sequential addition of nucleotides (N) beginningwith the 3'-terminal nucleotide of the desired nucleotide using themethod of this invention. After each reaction cycle of the PASS processthe growing oligonucleotide increases in length by at least oneoligonucleotide, and becomes the starting material for the next reactioncycle. As used herein the term can refer to either starting material orproduct and one of ordinary skill in the art will recognize what isintended by the term in a particular context.

Scheme 2 generally illustrates the method of this invention. A5'-protected monomer unit, such as phosphoramidite 7, is added to astarting material 8 in solution, in the presence of an activator, suchas tetrazole or preferably 4,5-dicyanoimidazole (DCI) (see U.S. patentapplication Ser. No. --, filed Oct. 15, 1996, entitled "ImprovedCoupling Activators for Oligonucleotide Synthesis"), to yield a product9 to which one nucleotide has been added via a phosphite triesterlinkage. As depicted in this figure the starting material 8 is a5'-deprotected otherwise protected oligonucleotide of length n, whereinn is an integer between 1 and 1000, and the product is a 5'-protectedoligonucleotide of length n+1. The 5'-deprotected oligonucleotidestarting material 8 is not anchored to a solid support, but rather,using standard methods, is simply 2', 3'-protected by protecting groups,such as base labile groups, that are compatible with the reaction of the5'-protected monomer units with the starting material and with5'-deprotection reactions. The elimination of 3'-anchoring to a solidsupport enhances the scope of the 3'-modifications that can beincorporated into oligonucleotides. Additionally, the 3'-terminalnucleotide no longer has the requirement of bearing the hydroxylsubstituent needed for support anchoring. Thus, modifications can beintroduced to the 3'-terminus which are not possible by solid phasesynthesis. This includes, but is not limited to, the use of polyethyleneglycol mono-methylether (molecular weight 5,000 to 100,000) or otherhigh molecular weight non-immunogenic units as the 3'-terminal monomerfor preparation of oligonucleotides with improved pharmacokineticproperties. (See U.S. patent application Ser. No. 08/434,465, filed May4, 1995, entitled "Nucleic Acid Ligand Complexes," which is incorporatedherein by reference).

After completion of the reaction between the 5'-protected monomer unit 7and starting material 8, the reaction mixture contains three species:unreacted 5'-protected monomer unit 7, unreacted starting material 8,and the product of the reaction, compound 9, which is a 5'-protectedoligonucleotide of length n+1. As discussed above any of the startingmaterial 8 (a 5'-deprotected oligonucleotide of length n) which fails toreact with the 5'-protected monomer unit 7 is referred to as the failuresequence, as this sequence was not extended. The product of thereaction, compound 9, is a 5'-protected oligonucleotide chain extendedby one nucleotide (length n+1), by the covalent reaction of the5'-hydroxy group of starting material 8, an oligonucleotide of length nwith the 3'-phosphoramidite group of the 5'-protected monomer unit 7.The product, compound 9, is the major component and the 5'-protectedmonomer unit 7 and the starting material 8 that did not react arepresent only in minor amounts.

At this stage of the process it is necessary to remove the unreacted5'-protected monomer unit from the reaction mixture, both to purify thematerials and to recover the monomer starting material. According tothis embodiment, non-reacted monomer is reacted to form an easilyremovable ionic species. Oxidation of the phosphite triester tophosphate triester may be carried out in the same reaction flask simplyby addition of an oxidizing agent. In situ oxidation gives the desiredoligonucleotide product 9, the phosphate salt 10 of monomer 7, as wellas unreacted oligonucleotide starting material 8. The monomer phosphatesalt 10 is the only free salt in the reaction mixture and thus is easilyremoved by techniques known to those in the art, including but notlimited to, filtration through an anion exchange resin or membrane orextraction with an aqueous phase. In an alternate variation of thisembodiment of the invention, the 3'-terminal monomer is a polyethyleneglycol mono-methylether of molecular weight 5,000 to 100,000, preferably20,000. In this case, a simple molecular weight cut-off membrane can beused to remove monomer 10.

After the unreacted monomer has been removed from the reaction mixture,the remaining filtrate may then be partitioned in any manner suitable toseparate the "oligonucleotide product" from the "failure sequence." Inone embodiment, the filtrate is applied to a material designed tointeract selectively or specifically with the 5'-protecting group (D-E),such as a reverse phase resin. The product is captured or retained onthe solid support by affinity of the 5'-protecting group constituent Dwith the resin. In a preferred embodiment, the filtrate is applied to amaterial designed to covalently react with the 5'-protecting group(D-E), such as a dienophile derivatized resin where D contains a dieneunit. The product is captured or retained on the solid support bycovalent reaction of the 5'-protecting group constituent D with theresin. The unreacted oligonucleotide starting material 8, which does notcarry the 5'-protecting group D, is washed away. The unreacted startingmaterial may be isolated and stored to be used as an intermediate in asubsequent synthesis. The retained oligonucleotide product 9 is thenreleased from the resin according to well known procedures. In certainembodiments, the oligonucleotide product is released by cleavage of thebond between the 5'-oxygen and the protecting group D-E. For example,when the 5'-protecting group is a trityl derivative, a reagent such asdilute dichloroacetic acid (DCA) may be used to cleave the trityl group,thereby releasing the oligonucleotide coupling product. The liberated5'-deprotected oligonucleotide coupling product 11 can then be used asthe starting material in an additional coupling reaction. ##STR8##

It is not a requirement of the present invention that the steps in themonomer addition cycle depicted in Scheme 2 occur in the exact sequencedescribed above. Alternatively, coupling and oxidation in situ can befollowed by covalent or affinity capture of the product and of monomer10 on a resin. Subsequent cleavage of the 5'-protecting group liberatesboth the product and the monomer. At this stage an extraction ormembrane-based filtration easily removes the unwanted monomer byproduct.

Utilization of the 5'-protecting group for anchoring of theoligonucleotide product allows for the possibility of using a widevariety of 3'-terminal modifications. These can be groups designed tofacilitate separation of the product of the reaction from the5'-protected monomer unit, such as a polymer of sufficient molecularweight to exploit molecular weight cut-off membranes for thisseparation, or a metal chelator to effect selective precipitation of theproduct. In such a case these groups contain a cleavable linker betweenthe 3'-terminus of the oligonucleotide and the modifying group, such asa succinate linker. Alternatively, non-nucleoside 3'-terminalsubstituents may enhance pharmacokinetic properties of oligonucleotideproducts, such as a polyethylene glycol mono-methylether or a distearylglycerol. (See U.S. patent application Ser. No. 08/434,465, filed May 4,1995, entitled "Nucleic Acid Ligand Complexes," which is incorporatedherein by reference). The 3'-terminal monomer may also serve as adetector for diagnostic applications of oligonucleotides, such as achelator designed to retain Tc99m for in vivo imaging. (See PatentApplication No. WO 96/02274, published Feb. 1, 1996, entitled"Conjugates Made of Metal Complexes and Oligonucleotides, AgentsContaining the Conjugates, Their Use in Radiodiagnosis as well asProcess for Their Production," which is incorporated herein byreference). In conventional solid phase oligonucleotide synthesis the3'-terminus is not accessible for introduction of such constituentssince it is utilized to anchor the growing chain to the solid support.

In contrast to the conventional solid phase synthesis process, theoligonucleotide product is preferably separated from unreacted startingmaterial each time a new coupling reaction is performed. Thus, the finaloligonucleotide product is obtained in essentially pure form and thecumbersome removal of highly homologous failure sequences is eliminated.Additionally, because the reaction is performed in the solution phase,the yields of the reaction of the monomer with the oligonucleotidestarting material are also significantly increased. Furthermore, acapping step becomes superfluous in this scheme, since only successfuloligonucleotide coupling products enter the next step of the process.The elimination of the capping step amounts to another efficiency gaincompared to the conventional process. The oligonucleotide startingmaterial that failed to undergo reaction with the 5'-protected monomerunit (failure sequence) is instead isolated and may be reused. Each timea failure sequence is reisolated during a PASS iteration, it can beblended into the starting material at the same step or iteration in asubsequent synthesis of the same oligomer, or of an oligomer that sharesthe same 3'-terminal fragment. (See Scheme 3). Failure sequences,therefore, become useful sequential building blocks for the subsequentmanufacture of oligonucleotides. This not only increases the efficiencyof the process, it also dramatically increases the purity of the finalcrude product. It further allows using the monomer as the limitingreagent and thus, dramatically increases process efficiency. ##STR9##The outlined synthetic scheme, which exploits the 5'-protecting group asthe anchor for separation of product from starting materials and allowsfailure sequences to become intermediates for subsequent syntheses, isnot limited to phosphoramidite coupling chemistry. It is compatible withother coupling reactions, such as, H-phosphonate or phosphate triestercoupling chemistry. (See Gaffney and Jones (1988) Tetrahedron Lett.29:2619-2622). This scheme also lends itself to automation ofoligonucleotide synthesis and is ideally suited for the large scalemanufacture of oligonucleotides with high efficiency.

Aspects of the technology described here have applications beyond thePASS synthesis process. For instance, the covalent capture of desired orunwanted species in oligonucleotide synthesis can also be applied to ahigh resolution, single-step purification method in conventional solidphase or solution phase processes. If only the terminal monomer bears adiene modified trityl group at its 5'-terminus, then selective anchoringof the full length product on a dienophile derivatized resin or membraneremoves all major failure sequences from the crude mixture. (See Scheme1). In another application, if a capping reagent is used which containsa moiety suitable for covalent capture (all D groups described aboveapply), such as a diene-modified acetic anhydride (or generally aD-modified acetic anhydride) or diene modified silyl chloride, such as3,5-hexadienoxyacetic anhydride or tri-(3,5-hexadienoxy)silyl chloride,then all capped failure sequences can be removed from a crudeoligonucleotide batch either after every monomer addition (in solutionphase oligonucleotide synthesis processes) or after cleavage of thecrude oligonucleotide from the solid support (in conventional solidphase oligonucleotide synthesis processes). In yet another application,the reaction of a diene modified trityl group with a dienophile modifiedresin allows facile preparation of cation exchange resins.

A dimer of 2'-fluoropyrimidine modified RNA oligonucleotides isassembled by the PASS process in Example 1 (Scheme 4). In the firstreaction phosphoramidite coupling chemistry is employed to form a3',3'-phosphodiester linkage. Oligonucleotides are often protectedagainst 3'- to 5'-exonucleolytic degradation by incorporation of a3',3'-phosphodiester linkage at the 3'-terminus. After coupling, thereaction mixture is oxidized in situ to produce unreacted thymidinestarting material 12, oxidized amidite monomer 15, and oxidized dimerproduct 14.

The oxidized amidite monomer 15 is removed by filtering the reactionmixture through a bed of diethylaminoethylene (DEAE) Sephadex®. HPLCanalysis of the filtrate indicates that the oxidized amidite monomer 15has been retained by the DEAE Sephadex® as shown in FIG. 3. Thefiltrate, which contains the oxidized dimer product 14 and the unreactedthymidine starting material 12, is concentrated and redissolved in 60%acetonitrile/water and loaded onto a C18 filter plug. The resin iswashed with 70% water/acetonitrile followed by 50% water/acetonitrile tofully elute the unreacted thymidine starting material 12. The resin,which now contains only the tritylated dimer product 14 is then washedwith water, followed by treatment with 80% acetic acid/water to effectdetritylation. The resin is then washed with 50% acetonitrile/water,which elutes the final product 16, while retaining the trityl species(FIG. 4). ##STR10##

Example 2 (Scheme 5) illustrates the method of this invention, whereinthe 5'-protected monomer unit is an H-phosphonate, rather than aphosphoramidite. In this example an H-phosphonate thymidine trimerbearing a 3',3'-internucleotidic linkage at the 3'-terminus(T-T-[3',3']-T trimer) 20 is prepared. The efficiency of the liquidphase coupling reaction was so high, that no unreacted 3'-terminalfragment 19 was detected. Thus, the reverse phase step is used only tocleave and separate the trityl group from the product.

Example 3 (Scheme 6) describes the synthesis of a phosphoramiditemonomer containing 5'-O-(4,4'-dioctadecyloxytrityl) (DOT) as the5'-protecting group (D-E).

Example 4 illustrates the ability to separate the coupling product fromthe unreacted oligonucleotide starting material (failure sequence) basedupon the selective or specific interaction of the 5'-protecting group(D-E) with a particular resin or phase. In this example, the mobility of4,4'-dioctadecyltriphenylmethanol (DOT) 23 on a C18 reverse phase resinis compared to that of 4-decyloxy-4'-methoxytritanol anddimethoxytritanol (DMT) (see Table 1). The strong interaction of the DOTgroup with C18 resin in organic solvents, such as methanol (R_(f) =0)and acetonitrile (R_(f) =0) enables the one-step separation of productfrom starting material by loading the mixture onto C18 resin and washingthe unreacted starting material away with an organic solvent. Thecoupled product can then be eluted from the chamber by cleavage of thetrityl protecting group with a haloacetic acid in an organic solvent.The trityl group is retained on the resin.

Example 5 describes the assembly of a hexamer oligonucleotide(5'-HO-T-T-A-C-T-[3',3']-T) in solution using an anion exchange mediumto remove the excess monomer and C18 reverse phase resin to selectivelycapture the 5'-DMT protected product while not retaining the failuresequence. As can be seen in Example 5, each monomer addition isaccomplished in two steps. In the first step phosphoramidite couplingchemistry is employed to couple the 5'-protected monomer unit to thestarting material. After coupling, the reaction mixture is oxidized insitu to produce unreacted starting material (failure sequence), oxidizedamidite monomer, and oxidized product. The oxidized amidite monomer isremoved by filtering the reaction mixture through an anion exchangemedium, such as, DEAE Sephadex®.

In the second step the filtrate, which contains the oxidized product andthe unreacted starting material (failure sequence), is treated with adilute acid to effect detritylation. Examples of dilute acids which canbe used include, but are not limited to, dilute mineral acids, dilutetrichloroacetic acid, dilute dichloroacetic acid (DCA), Lewis acids,such as, ZnBr₂, nitromethane, tosic acid and perchloric acid. Themixture is then separated by chromatography. Alternatively, the mixtureof product and unreacted starting material is first separated using areverse phase resin followed by detritylation to release thedetritylated product from the resin. The analytical data provided inExample 5 shows that the PASS process produces essentially pureoligonucleotide intermediates at every iteration with minimalconsumption of cost-limiting monomer.

Example 6 (FIG. 5) illustrates schematically an automatedextraction/filtration system 110 designed for use with the method ofthis invention, to separate the unreacted 5'-protected monomer unit fromthe remainder of the reaction mixture. As stated above, the method ofthis invention lends itself to automation and is thus ideally suited forlarge scale manufacture of oligonucleotides. The automatedextraction/filtration system 110 has two centers: an extraction vessel112 and a chromatography resin filtration chamber 114. The extractionvessel is in fluid communication with the chromatography resin chamberby a tube 118. A first three way valve 120 controls the flow of thecontents from the extraction vessel 112 into the chromatography chamber114. A second valve 122 controls the addition of solvents into chamber114. A third valve 124 controls the collection of effluent out ofchamber 114. All three valves are electronically coupled to a controller126, that provides signals that actuate all three valves 120, 122, and124 between their various flow positions.

Extraction vessel 112 is equipped with two inlet ports, 128 and 130, astirrer 132, and an outlet port 134. The reaction mixture is pumped intothe extraction vessel 112 through inlet port 128 and an extractionsolvent, such as CH₂ Cl₂, and an aqueous buffer are pumped into theextraction vessel through inlet port 130. The mixture may be agitatedwith stirrer 132, after which time the layers are allowed to separate.The first three way valve 120 is then opened and the bottom organiclayer flows through outlet port 134, into a conductivity monitor 136 andthen through tube 118 into chamber 114. The conductivity monitor iselectronically coupled to the controller 126. A rise in conductivityindicates that the organic layer has passed through the conductivitymonitor and the aqueous layer has begun to enter. The rise inconductivity is recognized by the controller 126 which sends a signal tothe first three-way valve 120 actuating the three-way valve to divertthe aqueous layer away from chamber 114.

Chamber 114 is equipped with three inlet ports 138, 140, and 142 and anoutlet port 144. The organic layer enters chamber 114 through inlet port138 and is pushed through the chamber 114 with a pressurized inert gassource, such as argon, which enters the chamber through inlet port 140.The chamber is then washed with solvent, i.e., CH₂ Cl₂, which enters thechamber through inlet port 142. The addition of solvent is controlled bythe controller which selectively actuates the second valve 122. Theorganic effluent is collected through outlet port 144 by opening of thethird valve 124 by the controller 126. The organic effluent contains theproduct of the reaction, which is the starting material extended by onenucleotide and unreacted oligonucleotide starting material (failuresequence). The unreacted 5'- protected monomer is retained in thechamber 114. After elution of the organic solvent, the chamber 114 iswashed with a buffered solution, added through inlet port 140, whichelutes the unreacted 5'-protected monomer unit. Chamber 112 is thenre-equilibrated with the organic solvent being used to elute thereaction mixture, i.e., CH₂ Cl₂. The organic effluent is next passedover a reverse phase resin, to separate the product from the unreactedoligonucleotide starting material (failure sequence). (See Example 6).

Example 7 describes the solution phase synthesis of the 15 baseoligonucleotide (5'-CTAAACGTAATGG-[3',3']-T-T-3') (SEQ ID NO: 1) usingpolyethylene glycol of 20,000 molecular weight as a 3' residuemodification. This example demonstrates the efficiency of solution phasesynthesis and the potential for preparing 3'-modified oligonucleotidesin solution which can not be directly prepared using conventional solidphase synthesis. This example outlines the basic steps required forsolution phase synthesis without the step wherein the oligonucleotidecoupling product is captured on a resin as in a typical PASS cycle.Thus, this example also demonstrates the impact on efficiency andproduct purity that product capture provides as envisioned in PASS. Withsuch product capture at each monomer addition cycle, cumbersomeprecipitations from diethyl ether are no longer necessary as inconventional solid phase synthesis. Additionally, because failuresequences are removed at each monomer addition cycle, the anion exchangechromatogram of the product obtained by PASS is expected to only show asingle product peak, rather than the multiple peaks present in thechromatogram of FIG. 6.

Example 8 (Schemes 7 and 8) describes the synthesis of various dienemodified trityl alcohols including a5'-di-(3,5-hexadienoxy)tritylthymidine phosphoramidite monomer (32) anda 5'-di-(2,4-hexadienoxy)tritylthymidine phosphoramidite monomer.

Example 9 (Scheme 9) demonstrates the use ofdienes--4,4'-di-3,5-hexadienoxytrityl alcohol (30) and4,4'-di-2,4-hexadienoxytrityl alcohol (36)--for efficient cycloadditionto maleimides (Reactions 1 and 2 respectively (Scheme 9)). Table 4 setsforth the reaction rates for these two reactions under variousconditions. From the data set forth in Table 4, it is clear thatmodified trityl compound (30) reacts faster under the various reactionconditions. It is also clear that, as expected, both the increase indienophile equivalents, as well as the addition of water to the reactionmixture increase the reaction rate. It is important to note reaction ofgreater than 50% of the diene substituents is sufficient for capture ofall the trityl alcohol or nucleotide on a maleimide-modified solid phasesupport, since there are two dienes present on each trityl group. Thisreduces the time needed for the reaction to take place.

For rate comparison purposes the Diels-Alder reaction was carried outwith 5'-O-(4,4'-di-3,5-hexadienoxytrityl)thymidine (5'-(DHDT)thymidine)(31) and with 5'-O-(4,4'-di-3,5-hexadienoxytrityl)thymidine3'-phosphoramidite (32) under the same reaction conditions given forreaction #93 (Table 4). The results are set forth in Table 5. Again,within 1 hour more than 50% of the diene groups underwent cycloaddition.This suggests that product capture, as envisioned in PASS, can occurwithin a reasonable time frame to allow rapid and efficient monomeraddition cycles. It is widely known that the rate of Diels-Aldercycloadditions can be tailored by using suitably substituted dienes anddienophiles. Thus, the product capture reaction rate can be tailored byemploying a suitable set of dienes and dienophiles.

Example 10 describes the preparation of 3'-PEG derivatizedoligonucleotides by PASS using the 4,4'-di-3,5-hexadienoxytritylprotecting group for capture of the oligonucleotide product on asubstituted maleimide-polystyrene resin. This capture step removes thenon-reacted starting oligonucleotide (failure sequence) from thereaction mixture. The latter can optionally be isolated and stored forblending into a subsequent production batch at the same point in theoligonucleotide assembly. A 3'-PEG terminal modification is useful interalia, for enhancing the pharmacokinetic behavior of therapeuticoligonucleotides in vivo.

Example 11 describes a general reaction scheme for the preparation ofnon-PEG derivatized oligonucleotides by Diels-Alder product captureusing a 5'-O-(4,4'-di-3,5-hexadienoxytrityl)-nucleoside(5'-O-DHDT-nucleoside) 46 as the diene and a maleimide substituted solidsupport 45 as the dienophile. (Scheme 11). As discussed above, thecapture of full length oligonucleotides on a resin or membrane isintegral to automating the PASS process. The general design of thecapture involves a trityl group or trityl analog being irreversiblybound to a solid support, such as a resin, membrane, or polymer 47. Oncebound, the oligonucleotide 49 is released by separating it from theirreversibly bound trityl group 48. An example of this is theDiels-Alder capture of the 5'-O-DHDT-nucleoside. Resin-bound activeDiels-Alder dienophiles covalently react with diene trityls andconventional methods of detritylation release the nucleoside from thesolid support and bound trityl group. This capture can be employed toprepare non-PEG derivatized oligonucleotides by PASS as described inExample 11 (Scheme 12). ##STR11##

A number of solid supports are envisioned to be suitable for capture andrelease using the Diels-Alder reaction. Preferred solid supports areinorganic oxides (silica, alumina, zeolites, controlled pore glass(CPG), etc.) that have surface hydroxyl groups that can be readilyfunctionalized. With the possible exception of CPG, these inorganicsolid supports often have a much higher loading capacity thancommercially available resins. Traditionally, these inorganic oxideshave been functionalized by silylating the hydroxyls with a silylatingagent that has a more versatile or reactive functional group. (Scheme13). ##STR12## Other methods of covalently linking the reactivedienophile are also envisioned, for example, esterification between amolecule such as 6-maleimido-caproic acid and the surface hydroxylgroup. (Scheme 14). Other covalent linkers between the surface anddienophile group may be used, if found to increase the surface loadingand/or reactivity of the dienophile. ##STR13##

Example 12 (Scheme 15) describes the preparation of a dimer usingproduct capture by Diels-Alder cycloaddition. The rate of capture of the3',3'-linked 5'-DHDTO-T-T dimer is dependent on the excess of resinbound maleimide groups. Product capture proceeds quantitatively. Thecaptured product is easily and quantitatively released from the resinwith 3% dichloroacetic acid in dichloromethane. After neutralization andconcentration, pure product is obtained.

Example 13 describes a method for assembly of oligonucleotides fromblocks by capturing one of the blocks on a resin using the cycloadditionof a 5'-O-(4,4di-3,5-hexadienoxytrityl) protected oligonucleotide to adienophile derivatized resin.

Example 14 (FIG. 8) illustrates schematically an automatedextraction/filtration system 200 and process designed for the automatedpreparation of an oligonucleotide bearing a 3'-terminal polyethyleneglycol using covalent capture of the monomer addition product at everycycle, as described in Example 10. As discussed above, the PASS process,which consists of a controlled, sequential polymeriszation of nucleosidephosphoramidites, can be performed in automated fashion. Each monomeraddition consists of a sequence of chemical processing steps. Thissequence remains the same for each monomer addition (cycle). The onlyvariable from cycle to cycle is the nature of the monomer that is added.A typical oligonucleotide consists of 2 to 12 different monomers, whichare added typically more than once in a dedicated, programmablesequence.

As can be seen in FIG. 8, the automated extraction/filtration system 200has three centers: a reaction vessel 212, a filtration chamber214--which contains the dienophile modified solid support 215--and anultrafiltration membrane system 218.

Example 14 also lists various ultrafiltration membranes compatible withthe conditions required for the separation of a product oligonucleotideand excess monomer after release from the capture resin. Membranes areevaluated based on reagent/product adsorption, retention, andreactivity. The membranes set forth in Example 14 were found to besuitable, based on flux rates as affected by solvent, loss of productdue to adsorption, and finally by diffuse reluctance FTIR.

For purposes of illustration, the preparation of a 3'-terminal PEGoligonucleotide is described in Example 14, however, this automatedmethod of synthesis can be done with or without a macromolecule attachedto the oligonucleotide. In the latter case, the molecular weight cut-offmembrane may be replaced by a liquid/liquid extraction step, as depictedin FIG. 5.

Example 15 describes the synthesis of maleimide derivatized tritylgroups. As discussed above, an integral part of the PASS process is amethod of removing n-1 sequences. One approach, is oligonucleotidesynthesis using monomers containing a maleimide-modified trityl group.These trityl groups are susceptible to reaction with diene-modifiedresins allowing separation of n-1 by simple washing of the resinfollowed by detritylation to release the full-length oligonucleotide.

Example 16 describes the use of diene-modified capping reagents for theselective removal of failure sequences during solution phase synthesisand conventional solid phase synthesis. Typically, failure sequences arecapped with acetic anhydride. The capping reaction with acetic anhydrideproceeds rapidly and near quantitatively. Thus, diene modified analogsof acetic anhydride, such as, 3,5-hexadienoic acid anhydride (74) and3,5-hexadienoxyacetic anhydride (75) (Scheme 18) allows efficientcapping of failure sequences and also enables removal of the cappedfailure sequence by cycloaddition to a dienophile derivatized resin ormembrane at each cycle during solution phase synthesis as described inExample 7. The 5'-acetyl capping groups introduced during conventionalsolid phase synthesis are removed during the ammonia cleavage anddeprotection step. In order to utilize reagents 74 or 75 as cappingreagents in solid phase synthesis and as subsequent handles forselective removal of the failure sequences, the oligonucleotide must bebound to the support via a linker, such as described in Example 12,which is selectively cleavable under non-basic conditions.Alternatively, a capping reagent can be used which is not susceptible toremoval under the typical basic deprotection conditions used at the endof conventional solid phase synthesis.

The hexadienoxysilyl chlorides (76, 77 and 78), allow selective removalof the failure sequences once the crude oligonucleotide is cleaved fromthe support with ammonia. The silyl ether group is not removed underthese conditions. Thus, the hexadienoxysilyl capped failures can beremoved from the desired product by reaction with a dienophilederivatized resin or membrane. ##STR14##

FIG. 10 illustrates generally the method of this invention as applied tothe assembly of peptides. With reference to FIG. 10, an N-protectedamino acid monomer unit 77, is added to a peptide starting material 78in solution in the presence of an activator. R⁶ is a standard carboxyblocking group, a soluble polymer or a diagnostic detector. TheN-terminal protecting group A is derivatized with a moiety X, designedto selectively and covalently react with group Y on resin 80. Reactionof 77 with peptide starting material 78, bearing a free N-terminal aminogroup, by standard coupling methods results in formation of the extendedpeptide product 79, now carrying the N-terminal protecting group. Inaddition, the reaction mixture contains the unreacted peptide startingmaterial 78, as well as excess N-protected amino acid monomer 77 andcoupling reagents. The peptide product 79, along with the unreactedmonomer 77 are selectively captured onto a resin by reaction of group Xwith substituent Y on the resin to form the solid-support bound products81 and 82. Release of 81 and 82 from the resin produces theN-deprotected peptide product 83, together with N-deprotected amino acidmonomer. The latter, being a free amino acid most likely in betaineform, is easily removed by either precipitation, extraction, or membranefiltration. Alternatively, the amino acid monomer 77 may be removed bythese techniques prior to capture of the product on the resin. Theextended, deprotected peptide product 83 is then ready to undergoanother addition cycle to extend the peptide chain.

In batch mode production, the unreacted peptide starting material 78(failure sequence) can be re-isolated and used in subsequent cycle atthe appropriate monomer addition step. This allows maximal efficiency ofamino acid monomer conversion into productive product.

The N-terminal protecting group X-A is designed such that its chemicalcomposition and the X-Y capture reaction are compatible withconventional peptide synthesis steps. Group A can be selected from anyof the N-protecting groups known to those of ordinary skill in the art,(see. e.g., Bodansky (1984) in Principles of Peptide Synthesis (SpringerBerlag, Berlin)), including but not limited to urethanes, such as fmocand Boc, benzyl groups, acyl groups, or triphenylmethyl groups. Thesubstituent X is designed to react with substituent Y on the resin withhigh selectivity. It is selected from groups such as dienes, inparticular 3,5-hexadienoxy or sorbic amide, dienophiles, in particularmaleimide, alkynes, silylether protected diols and disulfides. Thecorresponding substituent Y is chosen to be a selective covalentreaction partner for substituent X, such as a dienophile, diene,mercaptane, or borate.

In addition to serving as an anchor for selective product capture ateach monomer addition step, the introduction of amino acid monomerscarrying an N-terminal X-A protecting group at selected addition stepsor at the final step of peptide preparation serves to affinity purifythe product prior to final deprotection via capture on a Y-derivatizedresin. This capture dramatically increases the efficiency of segmentcondensation and subsequent purification. With capture of the N-terminalsegment on the resin, its C-terminal protecting group is removed andwashed away without requiring laborious and yield-reducing work-up. Thenow free C-terminal carboxyl group is activated by a standard method andcoupled to the C-terminal segment, bearing a free N-terminal aminogroup. After successful segment condensation, the reagents are washedaway, the product peptide is released from the resin by selectivebreaking of the N-A bond by standard methods to yield essentially pure,side chain protected peptide.

Example 17 illustrates the preparation and use ofN-2,7-di(3,5-hexadienoxyacetyl)fmoc protected amino acid monomers forpeptide synthesis by PASS.

Example 18 illustrates the preparation and use of2,7-di(maleimido)fluorene-9-methylchloroformate for peptide synthesis byPASS.

Example 19 illustrates peptide assembly using hexadienoxy-Boc protectedamino acids by PASS.

Example 20 illustrates the preparation of peptide nucleic acids by PASS.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE 1 Preparation ofN-4-benzoyl-3'-(5'-tert-butyldimethylsilyl-3'-(2-cyanophosphoryl)thymidyl)-2'-fluorocytidine(16) (Scheme 4)

5'-tert-butyldimethylsilylthymidine 12 (5'-TBDMS-thymidine) (0.15 g,0.42 mmol) was dissolved in dry acetonitrile (10 mL) under an argonatmosphere. Cytidine amidite 13 was added (0.43 g, 0.50 mmol) followedby tetrazole (6.5 mL, 0.45 M in acetonitrile). After 15 minutes reversephase HPLC analysis (C18, 4.6×100 mm, Buffer A: 100 mM triethylammoniumacetate pH 7.5, Buffer B: acetonitrile, 0 to 80% B over 2.5 minutes) ofthe reaction mixture showed the presence of dimer (2.4 minutes) as wellas unreacted thymidine 12 (1.4 minutes) and hydrolyzed amidite monomer(2.1 minutes) (FIG. 1). The reaction mixture was oxidized in situ (10mL, 0.2 M iodine in water/pyridine). HPLC analysis after oxidationreveals the presence of pyridine (0.9 minutes), unreacted thymidine 12(1.4 minutes), oxidized amidite monomer 15 (1.8 minutes), and oxidizeddimer 14 (2.3 minutes) (FIG. 2).

After oxidation the reaction mixture was passed with acetonitrilethrough a bed of DEAE Sephadex® pre-equilibrated with acetonitrile. HPLCanalysis of the filtrate indicates retention of the oxidized amiditemonomer 15 as shown in FIG. 3. The filtrate was concentrated underreduced pressure and the solid was re-dissolved in 60%acetonitrile/water and loaded onto a C18 chamber pre-equilibrated with70% water/acetonitrile. The chamber was washed with 70%water/acetonitrile followed by 50% water/acetonitrile to fully elute theunreacted thymidine 12. The chamber was then washed with water andtreated with 80% acetic acid/water to effect detritylation. Followingdetritylation the chamber was washed with 50% acetonitrile/water toelute the final product 16 (m/e 922, product 16 plus triethylamine).HPLC analysis shows elution of the detritylated species 16 at 1.7minutes (FIG. 4). ESMS (Electrospray Mass Spectrometry) of 16: Calcd820.27 (M+); Found 922.2 (M+H+TEA). ³¹ P NMR (121 MHz, CDCl₃, H₃ PO₄external standard) δ -0.73, -1.93. The trityl species was retained onthe chamber.

EXAMPLE 2 Preparation of a H-phosphonate thymidine trimer(T-T-[3',3']-T) (20)

Assembly of a H-phosphonate thymidine trimer bearing a3',3'-internucleotidic linkage at the 3'-terminus was synthesized asoutlined in Scheme 5. ##STR15##

Coupling of 12 to 5'-dimethoxytritylthymidine 3'-H-phosphonate 17

To a solution of 17 (0.75 g, 1.05 mmol) in 1:1 acetonitrile:pyridine (42mL) under argon was added 12 (0.25 g, 0.7 mmol), followed by a solutionof pivaloyl chloride (0.26 mL, 2.1 mmol) in 95:5 acetonitrile:pyridine(8.4 mL). The reaction was stirred for 10 minutes, at which time reversephase HPLC analysis showed complete conversion of 12 to dimer 18. Themixture was then concentrated in vacuo, dissolved in CH₂ Cl₂, andextracted with 0.05 M triethylammonium bicarbonate. The methylenechloride layer was applied to a plug of DEAE Sephadex® on a Buechnerfunnel. Reverse phase HPLC analysis of the filtrate showed completeremoval of the unreacted monomer 17. Dimer 18 was isolated inquantitative yield, by evaporation of the filtrate and its structure wasconfirmed by NMR and ESMS analysis. Unreacted monomer 17 was recoveredfrom the DEAE Sephadex® plug by washing with 1 M triethylammoniumbicarbonate. ESMS of 18: Calcd 946.4 (M+); Found 946.3. ¹ H NMR (300MHz, CD₃ CN) δ 9.21 (s, 2H), 7.45-7.24 (m, 11H), 6.94 (d, 1H, J=717.2Hz), 6.89-6.85 (m, 4H), 6.31-6.19 (m, 2H), 5.22-5.19 (m, 1H), 5.05-5.00(m, 1H), 4.22-4.19 (m, 1H), 4.11-4.10 (m, 1H), 3.81-3.80 (m, 2H), 3.75(s, 6H), 3.36-3.35 (m, 2H), 2.52-2.17 (m, 4H), 1.83 (s, 3H), 1.47 (s,3H), 0.91 (s, 9H), 0.10 (s, 6H). ³¹ P NMR (121 MHz, CD₃ CN) δ 14.03 (d),13.88 (d).

Detritylation of dimer 18

Dimer 18 (0.85 g, 0.9 mmol) was dissolved in methylene chloridesaturated with ZnBr₂ (10 mL, approximately 0.1 M ZnBr₂). After 15minutes reverse phase HPLC analysis showed complete detritylation. Thereaction was quenched with an equal volume of 1 M ammonium acetate. Theorganic layer was concentrated, the residue dissolved in 1:1acetonitrile:water and passed over a C18 plug on a Buechner funnel.Evaporation of the filtrate gave 0.29 g (50 % yield) of pure dimer 19.ESMS of 19: Calcd 644.2 (M+); Found 645.3. ¹ H NMR (300 MHz, CDCl₃) δ10.0, 9.85, 9.55, 9.45 (4s, 2H), 7.59-7.45 (m, 2H), 7.01 (d, 1H, J=712.3Hz), 6.39-6.19 (m, 2H), 5.35-5.23 (m, 1H), 5.14-5.03 (m, 1H), 4.31-4.22(m, 2H), 3.88-3.79 (m, 4H), 2.67-2.48 (m, 3H), 2.21-2.12 (m, 1H),1.89-1.88 (2 bs, 6H), 0.90 (s, 9H), 0.11 (s, 6H). ³¹ P NMR (121 MHz,CDCl₃)δ 8.45 (d), 8.30 (d).

Preparation of Trimer 20

To a solution of dimer 19 (0.25 g, 0.39 mmol) in 1:1 pyridineacetonitrile (23 mL) was added 17 (0.41 g, 0.58 mnmol), followed by asolution of pivaloyl chloride (0.14 mL, 1.16 mmol) in 95:5acetonitrile:pyridine (4.5 mL). The reaction was stirred under an argonatmosphere for 10 minutes, at which point HPLC analysis indicatedcomplete conversion of dimer 19 to trimer 20. The mixture was evaporatedto dryness, dissolved in CH₂ C₂, washed with 0.05 M triethylammoniumbicarbonate, and the organic layer was applied to a DEAE Sephadex® plugon a Buechner funnel. The filtrate was evaporated to give 20 inquantitative yield. ESMS of 20: Calcd 1234.4 (M+); Found 933.5 (M+H+withloss of DMT). ¹ H NMR (300 MHz, CD₃ CN) δ 9.34-9.27 (m, 2H), 8.58-8.56(m, 2H), 8.18-8.11 (m, 1H), 7.76-7.70 (m, 1H), 7.43-7.41 (m, 4H),7.35-7.23 (m, 13H), 6.88-6.84 (m, 4H), 6.26-6.15 (m, 3H), 5.78-5.71 (m,1H), 5.22-5.20 (m, 1H) 5.11-5.05 (m, 2H), 4.29-4.26 (m, 2H) 4.24-4.19(m, 2H), 3.85-3.84 (m, 2H), 3.76 (s, 6H), 3.74-3.72 (m, 1H), 3.38-3.28(m, 2H), 2.52-2.20 (m, 6H), 1.82 (s, 3H), 1.78 (s, 3H), 1.47-1.44 (m,3H), 0.90 (s, 9H), 0.11 (s, 6H). ³¹ P NMR (121 MHz, CD₃ CN) δ 15.86 (s),15.08 (s), 14.36 (s).

EXAMPLE 3 Preparation of 5'-O-(4,4'-dioctadecyltriphenylmethyl)thymidine-3'-O-(N,N-diisopropyl-2-cyanoethylphosphoramidite (26)

Assembly of a phosphoramidite monomer containing4,4'-dioctadecyltriphenylmethanol (DOT) as the 5'-protecting group (D-E)is illustrated in Scheme 6. ##STR16##

4,4'-Dioctadecyloxy-benzophenone (22)

Sodium metal (0.46 g, 20 mmol) was dissolved in ethanol (50 mL) and4,4'-dihydroxybenzophenone (1.0 g, 4.67 mmol) was added followed by1-bromooctadecane (7.8 g, 23.4 mmol) and a catalytic amount of sodiumiodide (approximately 30 mg) and the reaction mixture was refluxed for48 hours. The resulting suspension was cooled and filtered. The solidwas washed with dichloromethane followed by hexane and the white solidwas dried to afford compound 22 (2.85 g, 84.8% yield). ¹ H NMR (300 MHz,pyridine-d₅) δ 7.95 (d, J=8.7 Hz, 4H, aryl), 7.09 (d, J=8.7 Hz, 4H,aryl), 4.05 (t, J=6.6 Hz, 4H, 2×OCH₂), 1.80 (tt, J=6.6 and 7.5 Hz, 4H),1.48 (m, 4H), 1.33 (brS, 60H), 0.87 (t, J=6.6 Hz, 6H, 2×CH₃).

4,4'-Dioctadecyltriphenylmethanol (23)

To a suspension of benzophenone 22 (0.3 g, 0.42 mmol) in anhydrous THF(4 mL) was added phenylmagnesium bromide (0.55 mL, 1.0 M solution inTHF, 0.55 mmol) and the reaction was refluxed for 3 hours. An additionalamount of phenylmagnesium bromide (0.2 mL) was added and the heating wascontinued for 0.5 hours at which time all of the starting material haddissolved. The reaction was then cooled and 0.5 M HCl was added. Thesuspension was filtered and the solid washed with water (3×), hexane(2×) and dichloromethane (2×). The organic washes were pooled, dried(MgSO₄) and evaporated to afford 23 (0.21 g, 63.6% yield) as a whitesolid. ¹ H NMR (300 MHz, pyridine-d₅) δ 8.13 (brS, 1H, aryl), 7.81 (d,J=7.1 Hz, 2H), 7.7 (d, J=8.8 Hz, 4H), 7.42 (t, J=7.7 Hz, 2H), 7.34 (t,J=7.1 Hz, 1H), 7.07 (d, J=8.9 Hz, 4H), 3.98 (t, J=6.4 Hz, 4H), 1.77 (tt,J=7.9 and 6.5 Hz, 4H), 1.45 (m, 4H), 1.3 (brS, 60H), 0.87 (t, J=6.9 Hz,6H).

5'-O-(4,4'-Dioctadecyltriphenylmethyl)thymidine (25)

Compound 23 (2.1 g, 2.63 mmol) was coevaporated twice with toluene thendissolved in toluene (30 mL). Acetyl chloride (11 mL, 154.7 mmol) wasadded and the reaction was refluxed for 3 hours and then evaporated. Theresidue was coevaporated twice with toluene to afford crude 24. To 24was added pyridine (30 mL), DMAP (25 mg) and thymidine (0.45 g, 1.86mmol) and the reaction was stirred at room temperature overnight. Thesolvent was evaporated under reduced pressure and the residue was takenup in dichloromethane and washed with 5% sodium bicarbonate. The organicphase was dried (MgSO₄) and evaporated and the residue was purified onsilica gel (ethyl acetate/2% triethylamine) to afford after evaporationof the appropriate fractions compound 25 (DOT thymidine) (1.6 g, 84%yield) as pale yellow solid. ¹ H NMR (300 MHz, CDCl₃) δ 8.44 (brS, 1H,NH), 7.60 (s, 1H, H-6), 7.41-7.20 and 6.82 (m, 13H, DOT), 6.42 (t, J=6.1Hz, 1H, H-1'), 4.57 (m, 1H, H-3'), 4.05 (m, 1H, H-4'), 3.92 (t, J=6.5Hz, 4H, 2×OCH₃), 3.47 and 3.37 (ABX, 2H, H-5'), 2.38 (m, 2H, H-2'), 2.22(m, 1H, 3'-OH), 1.75 (m, 4H, DOT), 1.46 (m, 7H, 5-CH₃, DOT), 1.25 (brS,60H, DOT), 0.87 (t, 6H, 2×CH₃).

5'-O-(4,4'-Dioctadecyltriphenylmethyl)thymidine-3'--O--(N,N-diisopropyl-2-cyanoethylphosphoramidite(26)

DOT thymidine 25 was dissolved in dichloromethane (5 mL) anddiisopropylethylamine (0.3 mL, 1.75 mmol) and 2-cyanoethylN,N-diisopropylchlorophosphoramidite (0.15 mL, 0.63 mmol) was added withice bath cooling. The ice bath was removed and the reaction was stirredat room temperature for 4 hours at which point additional 2-cyanoethylN,N-diisopropylchlorophosphoramidite (0.1 mL) was added and the reactionwas stirred for 16 hours at room temperature. The reaction solution wasdiluted with dichloromethane and washed with 5% sodium bicarbonate, theorganic phase was dried (MgSO₄) and evaporated. The residue was purifiedon silica eluting first with hexanes, followed by 20% ethylacetate/hexanes all containing 2% triethylamine to afford 26 as resolveddiastereomers (fast 0.1 g, slow 0.18 g, 46.7 % yield). 26a (fastdiastereomer) ¹ H NMR (300 MHz, CDCl₃) δ 8.15 (br, 1H, NH), 7.91 (s, 1H,H-6), 7.63, 7.38-7.21, 6.82 (m, 13H, aryl), 6.39 (t, J=7.4 Hz, H-1'),4.67 (m, 1H, H-3'), 4.18 (m, 1H, H-4'), 3.92 (t, J=6.5 Hz, 2×OCH₃), 3.63(m, 4H), 3.51 and 3.33 (ABX, 2H, H-5'), 2.42 and 2.26 (m, 4H, H-2', CH₂CN), 1.73 (m, 4H), 1.41 (m, 4H), 1.25 (s, 60H), 1.16 (dd, J=2.7, 6.9 Hz,12H), 0.87 (t, J=6.9 Hz, 6H). ³¹ P NMR (121 MHz, CDCl₃) δ 150.64. 26b(slower diastereomer) ¹ H NMR (300 MHz, CDCl₃) δ 8.25 (br, 1H, NH), 7.91(s, 1H, H-5), 7.41 and 7.31-7.20 and 6.81 (m, 13H, aryl), 6.42 (dd, J=8Hz, H-1'), 4.67 (m, 1H, H-3'), 4.14 (m, 1H, H-4'), 3.92 (t, J=6.5 Hz,4H, 2×OCH₂), 3.82 and 3.76 (m, 2H), 3.54 (m, 2H), 3.47 and 3.31 (ABX,2H, H-5'), 2.62 (t, J=6.3 Hz, 2H, CH₂ CN), 2.54 and 2.34 (m, 2H, H-2'),1.76 (m, 4H), 1.25 (m, 60H), 1.16 and 1.05 (d, J=6.9 Hz, 12H, isopropylCH₃), 0.87 (t, J=6.6 Hz, 6H). ³¹ P NMR (121 MHz, CDCl₃) δ 150.23.

EXAMPLE 4 Resolution of alkyl substituted trityl groups on reverse phaseresin

The alcohols of 4,4'-dioctadecyltriphenylmethanol (DOT),4-decyloxy-4'-methoxytritanol, and dimethoxytritanol (DMT) were spottedonto a C18 reverse phase TLC plate and the plate was developed in threedifferent solvents (Table 1). As can be seen in Table 1, there is astrong interaction of the DOT group with the C18 resin in organicsolvents, such as methanol (R_(f) =0) and acetonitrile (R_(f) =0). Thisinteraction enables the one-step separation of the coupled product fromstarting material based upon the affinity or interaction of the tritylprotecting group for C18 reverse phase resin.

EXAMPLE 5 Preparation of 5'-HO-T-T-A-C-T-[3',3'-]-T-3' PASS usinghydrophobic affinity for product capture Preparation of5'-HO-T-[3',3']-T

5'-TBDPS-thymidine 12 (0.99 g, 2.07 mmol) was co-evaporated with drymethylene chloride and dissolved in 10 mL of dry methylene chloride.Thymidine amidite (2.0 g, 2.69 mmol) was added followed by tetrazole 0.5M in acetonitrile (21 mL, 10.5 mmol) and the reaction was stirred underargon. After 90 minutes, a solution of iodine/water/pyridine (0.2 M) wasadded until the dark brown color persisted, followed by 5% NaHSO₃ untilthe color returned to yellow. The concentrated reaction was partitioned(CH₂ Cl₂ /water) and the organic layer was dried with MgSO₄ andevaporated to dryness. The solid residue was dissolved inmethanol/minimal methylene chloride and pipetted onto a 75 g bed of DEAESephadex® equilibrated with water then methanol. The DEAE Sephadex® waswashed with 300 mL methanol and the combined methanol washes wereconcentrated to afford 2.42 g of a white foam.

Detritylation: The white foam was dissolved in 50 mL of 3% DCA andstirred at room temperature for 35 minutes, and then poured over 80 mLof silica gel equilibrated with methylene chloride. The gel was washedwith 150 mL of 3% DCA, followed by solutions from 100% methylenechloride through 6% methanol in methylene chloride. Appropriatefractions were combined and concentrated to give 1.58 g of detritylateddimer (5'-HO-T-[3',3']-T) in 90% yield for the two step process.

Preparation of the 5'-HO-C-T[3',3']-T

The 5'-HO-T-[3',3']-T dimer (1.47 g, 1.76 mmol) was dried under highvacuum overnight, and then co-evaporated with dry CH₂ Cl₂ and dissolvedin 8.5 mL of dry CH₂ Cl₂. Cytidine amidite (1.90 g, 2.28 mmol) was addedfollowed by tetrazole (0.5 M) in acetonitrile (17.6 mL, 8.78 mmol) andthe reaction was stirred under argon. After 50 minutes, a 0.5 M iodinesolution was added, followed by 5% NaHSO₃, changing the color from brownto yellow as described above. The concentrated reaction was partitioned(CH₂ Cl₂ /water) and the organic layer was dried (MgSO₄) and evaporatedto dryness. The solid residue was dissolved in methanol/minimalmethylene chloride and pipetted onto a 75 g bed of DEAE Sephadex®pre-equilibrated with water and then methanol. The DEAE Sephadex® waswashed slowly with methylene chloride and methanol and the combinedwashes were concentrated to afford 2.53 g of a yellow foam.

Detritylation: The foam was stirred in 50 mL of 3% DCA at roomtemperature. After 2 hours, the reaction mixture was pipetted onto an 80mL bed of silica gel pre-equilibrated with methylene chloride. Themixture was eluted with 3% DCA, followed by solutions from 100% CH₂ Cl₂through 6% methanol in CH₂ Cl₂. The appropriate fractions were combinedand concentrated to give 1.43 g of the detritylated trimer(5'-HO-C-T-[3',3']-T), 64% yield for the two step process.

Preparation of 5'-HO-A-C-T-[3',3']-T

The detritylated trimer 5'-HO-C-T-[3',3']-T (1.43 g, 1.1 mmol) was driedunder high vacuum overnight, coevaporated with dry methylene chlorideand dissolved in 6 mL of dry methylene chloride. Adenine amidite (1.24g, 1.45 mmol) was added, followed by 0.5 M tetrazole in acetonitrile (11mL, 5.57 mmol) and the reaction was stirred under argon. Afterapproximately 60 minutes, a 0.5 M iodine solution was added until thedark color persisted. The mixture was then stirred for 1 hour andconcentrated. The gum was partitioned (CH₂ Cl₂ /water) and the combinedorganic layer was dried (MgSO₄) and concentrated to yield 2.46 g of ayellow solid. The detritylation was carried out without DEAE Sephadex®purification.

Detritylation: The foam was stirred in 50 mL 3% DCA at room temperature,then pipetted onto a silica bed (approximately 120 mL) equilibrated withmethylene chloride. The reaction mixture was eluted with 3% DCA then100% methylene chloride through 10% methanol in methylene chloride. Theappropriate fractions were combined and concentrated to give 1.41 g ofthe detritylated tetramer (5'-HO-A-C-T-[3',3']-T), 72% overall yield forthe two step process.

Preparation of 5'-HO-T-A-C-T-[3',3']-T

The detritylated tetramer 5'-HO-A-C-T-[3',3']-T (1.41 g, 0.8 mmol) wasdried on high vacuum, then co-evaporated with dry methylene chloride anddissolved in 4.5 mL dry methylene chloride. Thymidine amidite (0.78 g,1.05 mmol) was added followed by tetrazole (0.5 M) in acetonitrile (8mL, 4.02 mmol) and the reaction stirred under argon. After 2 hours, a0.5 M iodine solution was added until the dark color persisted. Thereaction was then concentrated and the gum was partitioned (CH₂ Cl₂/water) and the combined organic layers were dried (MgSO₄) andconcentrated to yield 2.1 g of a yellow foam, which was analyzed by massspectrometry and reverse phase HPLC prior to elution through DEAESephadex®. Reverse phase HPLC analysis of the crude reaction mixtureafter oxidation showed the presence of pentamer, as well as, unreactedtetramer (failure sequence) and hydrolyzed amidite monomer. ESMS (M-1)803.74×3.

The yellow foam was dissolved in minimal methylene chloride and loadedonto a DEAE Sephadex® bed equilibrated with water and then methanol. TheSephadex® was washed with methanol, methylene chloride and thenacetonitrile. The appropriate fractions were combined and concentratedto give 1.48 g of material.

Detritylation: The material was stirred in 40 mL 3% DCA at roomtemperature, and then pipetted onto a silica bed equilibrated withmethylene chloride. It was eluted with 3% DCA, followed by solutions of100% methylene chloride through 20% methanol in methylene chloride. Theappropriate fractions were combined and concentrated to give 0.98 g ofthe detritylated pentamer (5'-HO-T-A-C-T-[3',3']-T), 64% overall yieldfor the two-step process. The ³¹ P NMR and its integration, areconsistent with the product.

Preparation of 5'-HO-T-T-A-C-T-[3',3']-T

The detritylated pentamer 5'-HO-T-A-C-T-[3',3']-T (0.96 g, 0.46 mmol)was dried under high vacuum, then co-evaporated with dry methylenechloride and dissolved in 5 mL of dry methylene chloride. Thymidineamidite (0.44 g, 0.59 mmol) was added followed by tetrazole (0.5 M) inacetonitrile (4.5 mL, 2.27 mmol) and the reaction was stirred underargon. Since the solution was not homogenous, 2 mL of acetonitrile wasadded. After 2 hours, an additional 0.15 g of monomer was added and thereaction was stirred overnight. A 0.5 M iodine solution was added,followed by 5% NaHSO₃, changing the color from brown to yellow. Theconcentrated reaction was partitioned (CH₂ Cl₂ /water) and the organiclayer was dried (MgSO₄) and concentrated to yield 1.61 g of a yellowsolid which was analyzed by MS. ESMS (M-1) 1384.01×2.

The crude reaction mixture (1.48 g) was absorbed onto C18 resin andloaded onto a bed of C18 resin (approximately 125 g) which had beenequilibrated with acetonitrile, followed by 70% water/acetonitrile. Theresin was first washed with 1:1 water:acetonitrile to elute the monomer,followed by acetonitrile and methylene chloride to elute the hexamer.The appropriate fractions were combined and concentrated to give 0.83 g,(66% yield) of a solid.

Detritylation. The solid was stirred in 20 mL of 3% DCA at roomtemperature. Trihexylsilane (2 mL) was added and stirring was continued.Upon addition of hexane a solid formed which was washed withhexane/ether to give 0.5 g of pink solid. The ³¹ P NMR and itsintegration, are consistent with the product.

Dowex C1-form can be used to remove residual DCA from a solid sample.For example, a T-A phosphoramidite dimer was found by NMR to containapproximately 1.2 equivalents of DCA following detritylation and hexaneprecipitation. A sample of this dimer (0.3 g) was dissolved inacetonitrile (5 mL) and loaded onto a column of Dowex C1-form (15 g)which had been pre-equilibrated with acetonitrile. The liquid was eluteddropwise and the column was then washed with 35 mL of acetonitrile andconcentrated to yield 0.26 g of a white foam. A sample checked by NMRshows approximately 95% reduction of acid.

EXAMPLE 6 Automation of PASS using hydrophobic affinity to capture theproduct

After the coupling reaction, e.g., the reaction of 12 with 17 in Example2 (Scheme 5), the reaction mixture is pumped into extraction vessel 112,through inlet port 128 (FIG. 5). Triethylammonium bicarbonate buffer(TBK) (0.05 M) and CH₂ Cl₂ are added to the extraction vessel throughinlet port 130, and the mixture is stirred. The layers are allowed toseparate. After separation, valve 120 opens and the methylene chloridelayer passes through conductivity meter 136, and onto a DEAE Sephadex®plug 114. A rise in conductivity indicates that the CH₂ Cl₂ hascompletely passed through the conductivity meter and the aqueous layerhas now entered the meter. At this time, valve 120 automaticallyswitches to divert the aqueous layer away from the DEAE Sephadex® plug.The organic layer is pushed through the DEAE Sephadex® plug with argonwhich enters the chamber through inlet port 140. The DEAE Sephadex® plugis then washed with CH₂ Cl₂, which is added through inlet port 142,controlled by valve 122. The CH₂ Cl₂ effluent, which contains theoligonucleotide product and unreacted oligonucleotide starting material(failure sequence), is collected through outlet port 144, controlled byvalve 124. Upon complete elution of CH₂ Cl₂, the unreactedphosphoramidite monomer, which has been retained on the Sephadex® plug,is eluted with the 1 M TBK. The Sephadex® plug is then re-equilibratedwith CH₂ Cl₂.

The CH₂ Cl₂ eluent, is then passed through a reverse phase resin toseparate the coupled product from the failure sequence. The coupledproduct, which has a DMT group attached to its 5'-end, is retained onthe resin, and the failure sequence is eluted from the chamber. Theresin is then washed with, acidic dichloroacetic acid (3% in CH₂ Cl₂),which cleaves the DMT protecting group and releases the coupled productfrom the chamber. The coupled product is eluted into a pH bufferedsolution to prevent decomposition due to excessive exposure to acid. Theeluent is concentrated and the coupled product used as the startingmaterial in the next reaction cycle.

EXAMPLE 7 Preparation of a 3'-PEG anchored 15mer DNA by solution phasesynthesis

An oligonucleotide of sequence 5'-CTAAACGTAATGG-[3',3']-T-T-3'(SEQ IDNO:1) was prepared by liquid phase synthesis, using polyethylene glycol(PEG) of molecular weight 20,000 as the 3'-terminal modification.Polyethylene glycol allows facile precipitation of the growingoligonucleotide chain during the individual steps. This example outlinesthe basic steps required for solution phase synthesis without theincorporation of the capture of the oligonucleotide coupling productonto a resin as in a typical PASS cycle. Thus, this example demonstratesthe impact on efficiency and product purity, that product captureprovides as envisioned in PASS. With such product capture at eachmonomer addition cycle, the cumbersome precipitations from diethyl etherare no longer necessary. In addition, because failure sequences areremoved at each monomer addition cycle, the anion exchange chromatogramof the product obtained by PASS is expected to only show a singleproduct peak, rather than the multiple peaks seen in FIG. 6.

This example provides the general procedures followed for each monomeraddition cycle for the preparation of a 3'-PEG anchored oligonucleotideby solution phase synthesis without the incorporation of product captureas a means to separate product from failure sequence. All of thefollowing reactions were performed in a one-neck flask with aself-sealing septum at room temperature. Disposable plastic syringeswere used.

Detritylation: 5'-DMT-nucleoside 3'-O-PEG (5.0 g) (20 k, loading: 45μmol/g) was dissolved in 50 mL of a mixture of dichloroacetic acid (DCA)and trihexylsilane (6.4 mL, 80 equivalents) in CH₂ Cl₂. After 9 minutesthe detritylated 5'-HO-nucleoside 3'-O-PEG was precipitated with ether(2×), washed, filtered and dried under vacuum.

Coupling reaction: The 5'-HO-nucleoside 3'-O-PEG was coevaporated 3times with 20 mL of anhydrous acetonitrile and dried under high vacuumfor 30 minutes. The flask was flushed with argon and closed to the outeratmosphere. Through the septa was injected: 50 mL of anhydrousacetonitrile to dissolve the 5'-HO-nucleoside 3'-O-PEG, 4.5 mL (0.1 M,2.0 equivalents) of amidite in anhydrous acetonitrile and 1.4 mL (1.0 M,6.0 equivalents) of DCI in acetonitrile. The solution was stirred underargon for 25 minutes, then precipitated with ether and dried bycoevaporation with 20 mL of anhydrous acetonitrile.

Oxidation: The precipitate was dissolved in 50 mL of anhydrousacetonitrile, and 8 mL (0.1 M) of iodobenzene diacetate in acetonitrilewas injected and the reaction mixture was stirred for 8 minutes.

Capping reaction: Acetic anhydride (6 mL), 2,6-lutidine (6 mL) andN-methylimidazole (6 mL) were simultaneously injected to the abovesolution and the reaction mixture was stirred for another 5 minutes. Thecapped oligonucleotide-PEG polymer was precipitated from ether asdescribed above in the detritylation procedure.

Crystallization: The capped oligonucleotide-PEG polymer was purified bycrystallization from 500 mL of absolute ethanol (100 mL/g) at 60° C.

The monomer addition cycle protocol is summarized in Table 2. Thestepwise coupling efficiency for the preparation of the 3'-terminal 10base fragment (10mer) (CGTAATGG-[3',3']-T-T) of oligonucleotide (SEQ IDNO:2), is shown in Table 3. The anion exchange HPLC chromatogram of thecrude 15mer (5'-CTAAACGTAATGG-[3',3']-T-T-3' (SEQ ID NO:1) aftercleavage from the PEG and deprotection is shown in FIG. 6.

EXAMPLE 8 Preparation of diene modified trityl alcohols

Example 8 (Schemes 7 and 8) describes the synthesis of various dienemodified trityl alcohols including a5'-O-(4,4'-di-3,5-hexadienoxytrityl) thymidine 3'-phosphoramiditemonomer 32. ##STR17##

Preparation of 4,4'-di-3,5-hexadienoxybenzophenone (29)

To a solution of 3,5-hexadienol (27) (13.7 g, 140 mmol) (Martin et al.(1980) J. Am. Chem Soc. 102:5274-5279) in anhydrous THF (335 mL) wasadded 4,4'-dihydroxybenzophenone (28) (10.0 g, 46.7 mmol) andtriphenylphosphine (36.7 g, 140 mmol) followed by the slow addition ofdiethylazodicarbonate (DEAD) (22.0 mL, 140 mmol). The reaction mixturewas stirred under argon overnight and then evaporated to dryness undervacuum. A precipitation from dichloromethane-hexane was carried out toremove residual reagents. The filtrate was concentrated in vacuo andpurified by column chromatography (silica gel; hexanes/CH₂ Cl₂, 3/2) toafford an impure product which was triturated (Et₂ O/hexane, 1/1) togive 7.12 grams of compound 29. Further purification of the filtrate bycolumn chromatography (silica gel; hexane/CH₂ Cl₂, 3/2) afforded anadditional 5.96 grams of 29 to give a total of 13.08 g (75%) of compound29 as a white solid. ¹ H NMR (300 MHz, DMSO-d₆) δ 2.50-2.64 (m, 4H),4.16 (t, J=6.5 Hz, 4H), 5.05 (d, J=10.1 Hz, 2H), 5.18 (d, J=15.7 Hz,2H), 5.77-5.92 (m, 2H), 6.17-6.47 (m, 4H), 7.10 (d, J=8.6 Hz, 4H), 7.72(d, J=8.7 Hz, 4H).

Preparation of 4,4'-di-3,5-hexadienoxytrityl alcohol (30)

Compound 29 (5.96 g, 15.91 mmol) was dissolved in anhydrous THF (133 mL)with slight heating. Phenylmagnesium bromide (32 mL of a 1.0 M solutionin THF, 32 mmol) was added to the solution and the mixture was stirredat room temperature under argon for 5 hours and evaporated to drynessunder vacuum. The residue was redissolved in dichloromethane and washedwith a saturated solution of ammonium chloride, followed by water. Theorganic phase was dried (MgSO₄), concentrated in vacuo, and purified bycolumn chromatography (silica gel; hexane/CH₂ Cl₂, 1/9) to yield 4.45grams (62%) of compound 30 as a yellow oil. ¹ H NMR (300 MHz, DMSO-d₆)δ2.45-2.56 (m, 4H), 3.98 (t, J=6.6 Hz, 4H), 5.01 (dd, J=1.5, 9.9 Hz, 2H),5.14 (dd, J=1.5, 16.5 Hz, 2H), 5.73-5.87 (m, 2H), 6.12-6.41 (m, 4H),6.25 (s, 1H), 6.84 (d, J=6.9 Hz, 4H), 7.06 (d, J=7.8 Hz, 4H), 7.15-7.33(m, 5H).

Preparation of 5'-O-(4,4'-di-3,5-hexadienoxytrityl)thymidine (31)

Compound 30 (3.5 grams, 7.73 mmol) was coevaporated with toluene (2×)and then dissolved in anhydrous toluene (85 mL). Acetyl chloride (33 mL,464 mmol) was added to the solution and the reaction mixture was heatedto reflux and stirred under argon. After 4 hours the reaction mixturewas concentrated in vacuo and the crude product was coevaporated withpyridine and then dissolved in anhydrous pyridine (42 mL). Thymidine(1.5 grams, 6.18 mmol), which had been coevaporated with pyridine anddissolved in anhydrous pyridine (42 mL), was then added to the solutioncontaining the crude product. A catalytic amount ofdimethylaminopyrimidine (DMAP) was added and the reaction mixture wasstirred under argon overnight and the solvent was evaporated. Theresidue was redissolved in dichloromethane and washed with a 5% aqueoussolution of sodium bicarbonate followed by water. The organic phase wasdried (MgSO₄), evaporated and purified by column chromatography (silicagel; EtOAc/hexane, 1/1) to afford 3.53 grams (84%) of compound 31 as anoff-white solid. ¹ H NMR (300 MHz, CDCl₃) δ 1.47 (s, 3H), 2.22-2.46 (m,2H), 2.50-2.63 (m, 4H), 3.35-3.58 (m, 2H), 3.85-4.09 (m, 5H), 4.51-4.60(m, 1H), 5.02 (dd, J=1.5, 10.4 Hz, 2H), 5.14 (dd, J=1.5, 17.3 Hz, 2H),5.68-5.83 (m, 2H), 6.12-6.45 (m, 5H), 6.82 (d, J=9.0 Hz, 4H), 7.18-7.46(m, 9H), 7.58 (s, 1H), 8.44 (s, 1H); Anal. Calcd for C₄₁ H₄₄ N₂ O₇.2H₂ O(712.8384): C, 69.08; H, 6.79; H, 3.93. Found: C, 69.34; H, 6.44; N,3.91.

Preparation of 5'-O-(4,4'-di-3,5-hexadienoxytrityl)thymidine3'-phosphoramidite (32)

Compound 31 (3.0 grams, 4.43 mmol) was dissolved in anhydrousdichloromethane and diisopropylethylamine (2.7 mL; 15.5 mmol) was added.The solution was cooled to 0° C. and2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (2.0 mL, 8.86 mmol)was added. The reaction mixture was allowed to warm to room temperaturewith stirring under argon. After 4 hours the solution was diluted withdichloromethane and washed with a 5% aqueous solution of sodiumbicarbonate (2×). The organic phase was dried (MgSO₄), concentrated invacuo, and purified by column chromatography (silica gel; EtOAc/hexane,3/7) to afford 2.8 grams (72%) of compound 32 as a fluffy white solid. ¹H NMR (300 MHz, CDCl₃) δ 1.01-1.20 (m, 12H), 1.41 (s, 3H), 2.25-2.67 (m,8H), 3.26-4.22 (m, 11H), 4.60-4.65 (m, 1H), 5.02 (dd, J=1.5, 10.4 Hz,2H), 5.14 (dd, J=1.5, 17.3 Hz, 2H), 5.69-5.84 (m, 2H), 6.11-6.48 (m,5H), 6.82 (dd, J=3.2, 8.9 Hz, 4H), 7.16-7.43 (m, 9H), 7.62 (d, J=15.2Hz, 1H), 8.05 (bs, 1H); ³¹ P NMR (300 MHz, DMSO-d₆) 152.9, 152.4; Anal.Calcd for C₅₀ H₆₁ N₄ O₈ P₁ (877.0276): C, 68.48; H, 7.01; N, 6.39.Found: C, 68.48; H, 7.22; N, 6.33. ##STR18##

Preparation of 4,4'-di-2,4-hexadienoxybenzophenone (35)

4,4'-Difluorobenzophenone (34) (4.8 grams, 22 mmol) was dissolved inanhydrous DMF (1 liter). NaH (95%; 5.6 grams, 220 mmol) was added andthe solution was cooled to 0° C. 2,4-Hexadienol (5.8 mL, 51 mmol) wasslowly added to the solution and the reaction mixture was allowed towarm to room temperature with stirring under argon overnight. Thereaction mixture was concentrated in vacuo, dissolved indichloromethane, and washed with water. The organic phase was dried(MgSO₄) and concentrated and purified by column chromatography (silicagel; hexane/CH₂ Cl₂, 1/3) to afford 2.07 grams (25%) of compound 35 as awhite solid. ¹ H NMR (300 MHz, DMSO-d₆) δ 1.73 (d, J=6.6 Hz, 6H), 4.68(d, J=6.0 Hz, 4H), 5.69-5.84 (m, 4H), 6.05-6.19 (m, 2H), 6.31-6.45 (m,2H), 7.08 (d, J=9.0 Hz, 4), 7.68 (d, J=10.2 Hz, 4).

Preparation of 4,4'-di-2,4-hexadienoxytrityl alcohol (36)

Compound 35 (2.0 grams) was dissolved in THF (45 mL) and phenylmagnesiumbromide (1.0 M solution in THF; 10.6 mL, 10.6 mmol) was added to thesolution. The reaction mixture was stirred at room temperature for 3hours, and evaporated to dryness under vacuum. The residue wasredissolved in dichloromethane and washed with a saturated solution ofammonium chloride, followed by water. The organic phase was dried(MgSO₄), concentrated in vacuo and purified column chromatography(silica gel; hexane/CH₂ Cl₂, 1/9) to afford 1.84 grams (77%) of compound36 as a pale yellow solid. ¹ H NMR (300 MHz, DMSO-d₆) δ 1.73 (d, J=6.9Hz, 6H), 4.54 (d, J=6.0 Hz, 4H), 5.68-5.80 (m, 4H), 6.02, 6.11 (m, 2H),6.20-6.37 (m, 2H), 6.85 (d, J=6.9 Hz, 4H), 7.05 (d, J=7.0 Hz, 4H),7.14-7.32 (m, 5H); Anal. Calcd for C₃₁ H₃₂ O₃ (452.5920): C, 82.27; H,7.13; Found: C, 82.30; H, 7.11.

The 5'-di-(2,4-hexadienoxy)tritylthymidine phosphoramidite monomer canbe prepared from compound 36 using the same procedure described abovefor the preparation of the 5'-O-(4,4'-di-3,5-hexadienoxytrityl)thymidinephosphoramidite (32).

EXAMPLE 9 Diels-Alder cycloaddition of diene substituted trityl alcoholswith N-ethylmaleimide

Example 9 (Scheme 9) describes the Diels-Alder reaction of dienesubstituted trityl alcohols--4,4'-di-3,5-hexadienoxytrityl alcohol (30)and 4,4'-di-2,4-hexadienoxytrityl alcohol (36)--with N-ethyl maleimide(Reactions 1 and 2 respectively). Table 4 sets forth the reaction ratesfor these two reactions under various reaction conditions. ##STR19##

Diels-Alder reaction of 3,5-hexadienoxytrityl alcohol (30)--Reaction 1

Compound 30 (50 mg, 0.11 mmol) was dissolved in acetonitrile (0.75 mL)and water (0.75 mL). N-ethyl maleimide (N-Et maleimide) (138 mg, 1.1mmol) was added and the reaction mixture was stirred at roomtemperature. After 3 hours ¹ H NMR analysis of the crude reactionmixture showed that the reaction had gone to completion. The reactionmixture was concentrated and loaded onto a silica gel plugpre-equilibrated with dichloromethane. The excess N-ethyl maleimide waswashed off with dichloromethane and the product was eluted with 10%MeOH/CH₂ Cl₂. The solvent was concentrated under reduced pressure toafford 38 mg (59%) of compound 37. ¹ H NMR (300 MHz, DMSO-d⁶) δ 0.97 (t,J=7.2 Hz, 6H), 2.02-2.19 (m, 4H), 2.20-2.34 (m, 2H), 2.42-2.53 (m, 4H),3.13-3.24 (m, 4H), 3.28-3.39 (m, 4H), 4.11 (t, J=6.3 Hz, 4H), 5.70-5.86(m, 4H), 6.22 (s, 1H), 6.87 (d, J=9.0 Hz, 4H), 7.07 (d, J=9.0 Hz, 4H),7.15-7.24 (m, 5H).

Diels-Alder reaction of 2,4-hexadienoxytrityl alcohol (36)--Reaction 2

Compound 36 (60 mg, 0.13 mmol) was dissolved in acetonitrile (2.0 mL).N-ethyl maleimide (166 mg, 1.3 mmol) was added and the reaction mixturewas stirred at room temperature. After 24 hours ¹ H NMR analysis of thecrude reaction mixture showed the reaction had gone to completion. Thereaction mixture was concentrated and loaded onto a silica gel plugpre-equilibrated with dichloromethane. The excess N-ethyl maleimide waswashed off with dichloromethane and the product was eluted with 10%MeOH/CH₂ Cl₂ and concentrated under reduced pressure to yield 50 mg(54%) of compound 38. ¹ H NMR (300 MHz, DMSO-d₆) δ 0.95 (t, J=7.1 Hz,6H), 1.32 (d, J=7.2 Hz, 6H), 2.48 (bs, 2H), 2.74 (bs, 2H), 3.05-3.46 (m,8H), 4.26 (t, J=8.4 Hz, 2H), 4.50 (t, 8.4 Hz, 2H), 5.67-5.86 (m, 4H),6.27 (s, 1H), 6.88 (d, J=8.7 Hz, 4H), 7.11 (d, J=8.7 Hz, 4H), 7.16-7.35(m, 5H); Anal. Calcd for C₄₃ H₄₆ N₂ O₇.2H₂ O (738.8762): C, 69.90; H,6.82; N, 3.79. Found: C, 71.16; H, 6.71; N, 3.92.

EXAMPLE 10 Preparation of 3'-PEG-linked oligonucleotides using productcapture by Diels-Alder cycloaddition

Example 10 (Scheme 10) provide the general procedures to be followed foreach monomer addition cycle, for the preparation of a 3'-PEG anchoredoligonucleotide by solution phase synthesis using the Diels-Aldercycloaddition reaction for the covalent capture of the oligonucleotideproduct. ##STR20## Coupling Reaction: PEG-dT-OH (20 k, 2.36 g, 0.11mmol, loading: 46 μmol/g) was dissolved in 20 mL of dry acetonitrile(CH₃ CN) under an atmosphere of dry argon. To this solution was added5'-O-(4,4'-di-3,5-hexadienoxytrityl)thymidine 3'-phosphoramidite (32)(140 mg, 0.16 mmol), followed by DCI in CH₃ CN (0.65 mL, 1.0 M, 6.0equivalents). The reaction was stirred under an atmosphere of dry argonfor 25 minutes, after which 350 mL of dry Et₂ O was added to precipitateout the 20 k-PEG containing material. The solids were filtered andwashed with Et₂ O (2×100 mL) and dried under vacuum for 1 hour to yield2.3 g of a white solid (98% mass yield).

Oxidation: The white solid, which contains coupled product 39, unreactedphosphoramidite 32 and unreacted PEG-dT-OH, is redissolved in 20 mL CH₂Cl₂ and oxidized with iodobenzene diacetate in CH₃ CN (8.5 mL, 0.1 M,0.27 g). After stirring for 8 minutes, the reaction mixture containsunreacted PEG-dT-OH, oxidized amidite monomer 40 and the oxidizedoligomer 41. The reaction mixture is then treated with 350 mL of dry Et₂O to precipitate the 20 k-PEG containing material and the solids arefiltered and washed with 2×100 mL Et₂ O. After drying under vacuum for 1hour, a white solid is isolated which contain the unreacted PEG-dT-OHand the oligomer 41.

Diels-Alder Cycloaddition: The solids are redissolved in 20 mL of 50% H₂O/CH₃ CN and loaded onto 1.2 g (10 equivalents based on a maleimideloading of 0.4 mmol maleimide/g resin) of maleimide-functionalizedpolystyrene, which has been prewetted with 5 mL of 50% H₂ O/CH₃ CN. Thereaction is warmed to 45° C. for 1 hour under an atmosphere of argon. Itis expected that reverse-phase HPLC analysis of the supernatant liquidwill reveal that the 5'-protected oligomer 41 has been completelyconsumed. The maleimide-derivatized polystyrene 42 can then be filteredand washed with 10 mL of 50% H₂ O/CH₃ CN, to yield 3.5 g of3'-PEG-5'-DHDT Diels-Alder conjugate oligomer (42) as a solid resin.

Detritylation/Oligonucleotide Release: It is anticipated that the 3.5 gof Diels-Alder conjugate resin 42 (loading: 75 μmol/g) can be suspendedin 20 mL of CH₂ Cl₂. To this suspension is added a mixture of DCA andtrihexylsilane (6.4 mL, 80 equivalents) in CH₂ Cl₂. After 9 minutes thepolystyrene-maleimide resin (44) is removed via filtration. ThePEG-nucleoside (43) is then precipitated twice with Et₂ O (500 mL),washed, filtered and dried under vacuum. The resultant PEG-nucleoside isdeprotected at the 5'-position and is ready for the next couplingreaction of the sequence.

EXAMPLE 11 Preparation of non-PEG derivatized oligonucleotides byDiels-Alder product capture

Scheme 12 illustrates a general reaction scheme for the preparation of anon-PEG derivatized oligonucleotide by Diels-Alder product capture usinga 5'-O-(4,4-di-3,5-hexadienoxy trityl)nucleoside (5'-O-DHDT-nucleoside)as the diene and a maleimide substituted solid support as thedienophile. Briefly, the Diels-Alder capture PASS cycle is performed inthe following manner: A 3'-blocked oligomer is coupled in the usualfashion with a 5'-O-(4,4'-hexadienoxytrityl)nucleoside3'-phosphoramidite. The 3'-blocking group is a lipid or polysaccharide,or a more traditional solution-phase blocking group such as acetyl,pyranyl, or silyl group, such as tert-butyldiphenylsilyl ether.##STR21## Coupling/Oxidation/Capture Sequence. In CH₃ CN, theappropriate 3'-blocked oligonucleotide of length n (50) is coupled with2.0 equivalents of the amidite monomer 51 by treatment with a 1.0 Msolution of DCI in CH₃ CN. The coupling reaction takes less than 25minutes and is monitored by TLC. Upon completion of the couplingreaction, the solution is treated directly with 8.0 equivalents ofiodobenzene diacetate as a 0.1 M solution in CH₃ CN. The oxidationsequence is complete within 8 minutes and the crude reaction mixture isapplied directly to the solid support bearing the dienophile.Polystyrene, bearing a maleimide dienophile is the preferred solidsupport. The Diels-Alder cycloaddition reaction is accelerated byutilizing a solvent of 1:1 CH₃ CN:H₂ O. The oligonucleotide, nowcovalently bound to the solid support 52, is easily separated from theunreacted starting oligonucleotide 50 (failure sequence) and reagentsvia simple filtration and washing of the resin beads. The amiditemonomer 51 which also has a 5'-DHDT group is also bound to the resin(53).

Detritylation/Release Sequence. The washed and dried resin, bearing thecovalently bound oligonucleotide (52), as well as, unreacted monomerphosphate (53), is washed with a solution of 3% DCA/CH₂ Cl₂, elutinginto a neutralizing buffer to prevent acid-mediated decomposition of theoligonucleotide chain. The released oligomer (54) and monomer phosphate(55) are separated from one another via aqueous extraction. The productoligonucleotide in the organic phase is dried and exchanged intoacetonitrile by ultrafiltration.

EXAMPLE 12 Preparation of a Dimer using Product capture by Diels-Aldercycloaddition ##STR22## Preparation of 5'-DHDTO-T-[3',3']-T-OSiPDBT-5'(56)

5'-TBDPSiO-dT-3'-OH (12) (0.21 g, 0.43 mmol) was dissolved in 10 mLacetonitrile. 5'-DHDTO-dT phosphoramidite (32) (0.5 g, 0.52 mmol) wasadded to this solution followed by 3.0 mL of 1.0 M DCI in acetonitrile(3.0 mmol). This solution was stirred under argon for 20 minutes, atwhich time 11 mL of a solution of 0.2 M I₂ in pyridine/water was added.The oxidation reaction was allowed to proceed for 5 minutes and wasfiltered (4×) through DEAE Sephadex® to remove most of the yellow color.A yellow solid 56 (0.23 g) was isolated.

Product Capture: The Diels-Alder capture reaction was performed withvariation in the amount of polystyrene supported maleimide (PS-M) used,as follows: 10 eq, 5 eq, 2.5 eq, 1 eq. The procedure below for all ofthe reactions was as follows. The [3',3']-dT-dT-OTDHD dimer (11 μmol)(56) was dissolved in 400 μL of acetonitrile. This solution was added toa suspension of PS-M in 1.0 mL of 3/1 CH₃ CN/water and then warmed to65° C. The course of the reaction was monitored by TLC (2/1EtOAc/hexanes), by the disappearance of the reactant at R_(f) =0.15, andvia HPLC analysis (C18, 4.6×100 mm, Buffer A: 100 mM triethylammoniumacetate pH 7.5, Buffer B: acetonitrile, 0 to 80% B over 2.5 minutes).The % reaction was determined by comparison to the initial ratio ofdimerized material (2.65 min) to unreacted 5'-TBDPSiO-dT-3'-OH monomer(12) (1.71 min). (See FIG. 7). It is interesting to note that the linesdrawn for 2.5 equiv., 1.0 equiv., and control (No PS-M) all showreaction (disappearance of dimer) occurring after 4 hours. The reactionis not a Diels-Alder capture, but is rather decomposition of the dimervia what is believed to be hydrolysis. A new material at 1.47 minutesand 2.30 minutes appears in the HPLC traces. This material may be5'-TBDPSiO-dT-3'-phosphate (1.47 minutes) and 5'-DHDTO-dT-3'-phosphate.Since 5'-TBDPSiO-dT-3'-OH is also expected to be produced in the courseof the hydrolysis reaction, the relative rates of Diels-Alder capturecannot be directly obtained from these traces as the internal standardis not appropriate in the cases where hydrolysis is evident. Hydrolysiscan be corrected for by adjusting to the amount of5'-TBDPSiO-dT-3'-phosphate evident in the later traces. This process isnot significant in the case of 5.0 equiv. and 10.0 equivalents.

Release/Detritylization: 286 mg of PS-M derivatized with 11 μmol of the[3',3'] dimer 57 was suspended in 0.25 mL dichloromethane. To thissolution was added 2.6 mL of 3% DCA in dichloromethane. The PS-Mimmediately turned bright orange. The suspension was agitated for 5minutes, whereupon the dichloromethane solution was removed viafiltration from the PS-M. The solution obtained was immediately filteredthrough a pad of Dowex-Cl⁻ ion exchange resin with dichloromethane. Thefiltrate was then concentrated to yield 12 mg of a white, glassy solid(contains some residual solvent and aliphatic impurities). ¹ H NMR and³¹ P-NMR are consistent with the desired product compound 58.

EXAMPLE 13 Preparation of an oligonucleotide from two blocks usingfragment anchoring by Diels-Alder cycloadditon

The PASS oligonucleotide synthesis scheme allows facile and efficientpreparation of oligonucleotide blocks, which can be coupled to eachother in a modified PASS cycle as illustrated in Scheme 16. Briefly, theoligonucleotide block 59, prepared by PASS monomer addition cycles asoutlined above, is reacted with a maleimide resin to give the resinanchored oligonucleotide block 61. The 3'-terminal PEG is removed fromthis block by reductive cleavage of linker L with titanium trichloride,yielding resin bound fragment 63, which has a free 3'-terminus.Phosphitylation of 63 with N,N-diisopropyl-2-cyanoethyl-chlorophosphineresults in the 3'-terminal phosphoramidite 64. Compound 64 is thencoupled to oligonucleotide block 62, obtained from detritylation ofoligonucleotide block 60 after capture on a maleimide resin andsubsequent detritylation. The coupling reaction is followed by oxidationof the phosphite triester linkage to the corresponding phosphatetriester, followed by release of the product oligonucleotide from theresin with dichloroacetic acid, giving oligonucleotide fragment 60.##STR23##

EXAMPLE 14 Automation of PASS using Diels-Alder product capture forpreparation of oligonucleotides

The coupling reagents are added to reaction vessel 212 and reaction isallowed to proceed as described in Example 10. Upon completion of thecoupling reaction, the reaction mixture is circulated through the dieneor dienophile modified resin or membrane (hereafter referred to as thesupport), which is contained in vessel 214, to covalently capture theoligonucleotide. The time required for the capture step can becontrolled by monitoring the disappearance of the coupling product fromsolution either by an HPLC or in line UV assay (not shown). The supportis then rinsed to elute all failure sequences not containing the dieneor dienophile. Oxidation can be accomplished either after theoligonucleotide is attached to the support or in solution prior toattachment to the dienophile support. The oxidation solution must bethoroughly removed from the resin prior to detritylation. This removalis conveniently controlled by in-line conductance monitoring (notshown). The support is then rinsed with DCA/CH₂ Cl₂ to remove thegrowing oligomer and captured excess monomer from the resin, thusallowing the only species in the solution to be the 5'-deprotectedoligonucleotide and monomer in a DCA/CH₂ Cl₂ mixture. This mixture isthen brought into contact with a membrane separator (218) to remove theDCA and the excess monomer, in addition to a solvent exchange toacetonitrile. Alternatively, the monomer may be separated byprecipitation or extraction. The only species remaining in solution isthe macromolecule attached oligomer in acetonitrile. This solution isnow ready for the next coupling reaction.

The removal of all n-1 species with use of the dienophile support, thuseliminates the use of a capping step, and the solution is ready to beoxidized and or circulated through the dienophile support. Thedienophile support can contain a cleavable linker between the dienophilemoiety and the resin or membrane, such as an amide bond. This cleavablelinker allows facile regeneration of the dienophile support. Linkerssuch as these are well known to those skilled in the art.

Membrane Evaluation

Recovery of Pegylated Deoxythymidine after Exposure to a PolypropyleneUltrafiltration Membrane: Acetonitrile Solvent System. A solution of2.74 mM 20 k PEG-deoxythymidine (PEG-dT) was made by dissolving 1.49grams of 46 μmol dT/gram PEG-dT in 25 ml of acetonitrile. Aliquots (2mL) of the solution were then exposed to areas of 5.73 squarecentimeters of the working surface of a polypropylene ultrafiltrationmembrane (3M®) for periods of 0.25, 1 and 4 hours in 50 mL Falcon®tubes. The starting solution was rinsed from the Falcon® tubes andmembranes with two 25 mL washes of acetonitrile. The wash solvent wasassayed for PEG-dT spectrophotometrically by absorbance at 260 nm andbalanced relative to the absorbance of the starting PEG-dT. A control tomeasure losses to the tube and glassware was performed by exposing aFalcon® tube without a membrane to 2 mL of the starting solution for 4hours and assaying for PEG-dT at 260 nm. Results are shown in Table 6.

Recovery of Pegylated Deoxythymidine after Exposure to a PolypropyleneUltrafiltration Membrane: Methylene Chloride Solvent System. A solutionof 2.72 mM 20 k PEG-deoxythymidine (PEG-dT) was made by dissolving 1.48grams of 46 μmol dT/gram PEG-dT in 25 mL of methylene chloride. Aliquots(2 mL) of the solution were then exposed to areas of 5.73 squarecentimeters of the working surface of a polypropylene ultrafiltrationmembrane (3M®) for periods of 0.25, 1 and 4 hours in 50 mL Falcon®tubes. The starting solution was rinsed from the Falcon® tubes andmembranes with two 25 mL washes of methylene chloride. The wash solventwas assayed for PEG-dT spectrophotometrically by absorbance at 260 nmand balanced relative to the absorbance of the starting PEG-dT. Acontrol to measure losses to the tube and glassware was performed byexposing a Falcon® tube without a membrane to 2 mL of the startingsolution for 4 hours and assaying for PEG-dT at 260 nm. Results areshown in Table 7.

Recovery of Pegylated Deoxythymidine after Exposure to a RegeneratedCellulose Ultrafiltration Membrane: Acetonitrile Solvent System. Asolution of 2.85 mM 20 k PEG-deoxythymidine (PEG-dT) was made bydissolving 1.55 grams of 46 μmol dT/gram PEG-dT in 25 mL ofacetonitrile. Aliquots (2 mL) of solution were exposed to areas of 5.73square centimeters of the working surface of a polypropyleneultrafiltration membrane (Millipore^(a), 10KPLGC) for periods of 0.25,1, 4 and 24 hours in 50 mL Falcon® tubes. The starting solution wasrinsed from the Falcon® tubes and membranes with a 25 mL wash ofacetonitrile. The membrane was soaked in 25 mL of acetonitrile for sixdays and then washed with an additional 25 mL of acetonitrile. The washsolvents were assayed for PEG-dT spectrophotometrically by absorbance at260 nm and balanced relative to the absorbance of the starting PEG-dT. Acontrol to measure losses to the tube and glassware was performed byexposing a Falcon® tube without a membrane to 2 mL of the startingsolution for 4 hours and assaying for PEG-dT at 260 mn. Results areshown in Table 8.

Centrifugation of Pegylated Deoxthymidine in Acetonitrile/Ether: DiethylEther, Diisopropyl Ether and N-Butyl Ethers Compared. A solution of 2.34mM 20k PEG-deoxythymidine (PEG-dT) was made by dissolving 0.4855 gramsof 46 mol/gram PEG-dT in 10 ml of acetonitrile. Aliquots of 0.5, 0.25and 0.125 mL were precipitated by addition of 1 mL of either diethylether, diisopropyl ether or N-butyl ether. The precipitates werecentrifuged at approximately 4,400 times gravity for 2 minutes. ThePEG-dT content of the supernatants was determined spectrophotometricallyand balanced relative to the starting-PEG-dT. A control to show lossesto handling was performed by centrifuging and assaying 1.5 mL of thestarting solution by the above method. The results are summarized inFIG. 9.

Compatibility by Flux and FTIR evaluations. Polyvinylidienedifluroride(PVDF) and polypropylene membranes were evaluated by soaking them in thefollowing solvent systems: acetonitrile, methylene chloride, theCoupling/Capping/Oxidation (c/c/0) solution in acetonitrile, and themixture of 3% DCA in methylene chloride. Pieces of the membranes 11/2"in diameter were submersed in the solutions, allowed to soak for 24hours, placed into a membrane holder for flux evaluation of the initialsolution, and then rinsed with acetonitrile for further acetonitrileflux evaluations. Thus, the membrane sample was rinsed of any excessreagent that may have remained on the membrane after soaking insolution. The acetonitrile flux rates after exposure to the varioussolvents are listed in Table 9. As can be seen, there are only minorchanges in the flux rate in (mL/min/cm²) between the PVDF and thepolypropylene membranes.

In a retention study, the regenerated cellulose membrane was determinedto have retained some of the PEG, as measured by FTIR. The silicone,ceramic, polyolefin and HDPE membranes are under investigation.##STR24##

Preparation of 4,4'-di-(3-t-butyldimethylsilyloxypropoxy)-benzophenone(66)

4,4'-dihydroxybenzophenone (28) (10 g, 46.7 mmol) was reacted underMitsunobu conditions with t-butyldimethylsilyloxy-3-propanol (40 gcrude, approx. 150 mmol), DEAD (22.1 mL, 140.0 mmol) andtriphenylphosphine (36.7 g, 140.0 mmol) in dry tetrahydrofuran at 0° C.The reaction was allowed to warm to room temperature under argon. After24 hours the reaction was concentrated and the salts precipitated withhexane/ether and filtered. The remaining material was purified by columnchromatography column chromatography (silica gel; gradient of hexane to85% hexane/ethyl acetate) to afford to afford 14 g of the desiredproduct compound 66 in 54% yield. ¹ H NMR (300 MHz, CDCl₃) δ 7.92 (d,4H), 7.68 (d, 4H), 4.13 (t, 4H), 3.76 (t, 4H), 1.96-1.88 (m, 4H), 0.85(s, 18H), 0.02 (s, 12H).

Preparation of4,4'-di-(3-t-butyldimethylsilyloxypropoxy)-triphenylmethanol (67)

The protected benzophenone 66 (5.7 g, 10.2 mmol) was dissolved in 40 mLdry THF and phenylmagnesium bromide (20.5 mL, 20.4 mmol) was added. Thereaction was stirred under argon at room temperature for 2 hours,concentrated, partitioned between dichloromethane and saturated ammoniumchloride, and washed with water. The organic layer was dried (MgSO₄) andconcentrated to yield 6.5 g of a yellow gum, compound 67, inquantitative yield and used directly in the next step. ¹ H NMR (300 MHz,CDCl₃) δ 7.27-7.17, 7.05, 6.82 (m, 13H), 6.23 (s, 1H), 3.99 (t, 4H),3.73 (t, 4H), 1.91-1.83 (m, 4H), 0.84 (s, 18H), 0.02 (s, 12H).

Preparation of 4,4'-di-(3-hydroxypropoxy)-triphenylmethanol (68)

The trityl compound 67 (6.37 g, 10 mmol) was deprotected by treatmentwith triethylamine hydrofluoride (3.64 g, 30 mmol) in acetonitrile atroom temperature for 16 hours. The reaction was concentrated andpurified by column chromatography (silica, gradient: 1:1 hexane:ethylacetate to ethyl acetate:5% methanol all with 1% triethylamine)affording 2.8 g of the desired product 68 as a yellow gum in 69% yield.¹ H NMR (300 MHz, CDCl₃) δ 7.30-7.18, 7.06, 6.83 (m, 13H), 6.22 (s, 1H),4.55 (t, 2H), 4.07-3.98 (m, 4H), 3.57-3.52 (m, 4H), 1.88-1.80 (m, 4H).

Preparation of 4,4'-di-(3-p-toluenesulfonoxypropoxy)-triphenylmethanol(69)

A solution of tosyl chloride (1.43 g, 7.49 mmol) and 2,4,6-collidine (1mL, 7.49 mmol) in acetonitrile was added to compound 68 (1.39 g, 3.4mmol) in 15 mL acetonitrile. The reaction was stirred at roomtemperature under argon for 2.5 days and then concentrated. The residuewas purified by column chromatography (silica, 60% ethyl acetate inhexane with 1% triethylamine) to give 0.6 g of the tosylated compound 69in 25% yield. ¹ H NMR (300 MHz, CDCl₃) δ 7.76 (d, 4H), 7.38 (d, 4H),7.32-7.06, 7.01, 6.73 (m, 13H), 6.26 (s, 1H), 4.17 (t, 4H), 3.88 (t,4H), 2.33 (s, 6H), 2.04-1.96 (m, 4H).

Preparation of 4,4'-di-(3-azidopropoxy)-triphenylmethanol (70)

To a solution of 69 (0.6 g, 0.84 mmol) in 15 mL of dry DMF was addedlithium azide (0.12 g, 2.51 mmol). The reaction was stirred under argonat room temperature overnight, concentrated and purified by columnchromatography (silica, 60% ethyl acetate in hexane with 1%triethylamine) to yield 0.38 g (100%) of compound 70 as a yellow gum. ¹H NMR (300 MHz, CDCl₃) δ 7.34-7.17, 7.07, 6.85 (m, 13H), 6.25 (s, 1H),3.99 (t, 4H), 3.50 (t, 4H), 2.00-1.77 (m, 4H).

Preparation of 4,4'-di-(3-aminopropoxy)-triphenylmethanol (71)

The azide (70) (0.25 g, 0.55 mmol) was warmed with activated charcoal inmethanol, filtered and concentrated. The residue was again dissolved in50 mL of methanol and 55 mg 5% palladium on carbon was added. The flaskwas evacuated and a hydrogen filled balloon added. After 1 hour at roomtemperature the catalyst was filtered. The reaction was concentrated andused directly in the next step.

Preparation of 4,4'-di-(3-maleimidopropoxy)-triphenylmethanol (73)

The crude residue 71 was dissolved in 50 mL 1:1 acetonitrile:water andstirred in ice bath. Methoxy carbonyl maleimide reagent (72) (0.16 g,0.98 mmol) was added and over 2 hours the pH was observed to drop from10.1 to 5. The pH was then adjusted to 2 with 1 M sulfuric acid and thereaction concentrated. The residue was partitioned between ethyl acetateand brine. The organic layer was concentrated, re-dissolved in 1:1acetonitrile:water and stirred with 10 mL 5% sodium bicarbonate. After17 minutes the reaction was acidified to pH 3 with 1 M sulfuric acid.Ethyl acetate (20 mL) was added and the solution was partitioned and theaqueous layer back extracted with ethyl acetate. The combined organiclayers were concentrated and purified by column chromatography (silica,ethyl acetate and hexane mixtures) to give 0.104 g of product 73 in 36%yield. ¹ H NMR (300 MHz, CDCl₃) δ 7.30-7.18, 7.04, 7.68 (m, 13H), 7.02(s, 4H), 6.24 (s, 1H), 3.92 (t, 4H), 3.57 (t, 4H), 1.99-1.89 (m, 4H). MS(MS+566). Anal. Calcd. for C₃₃ H₃₀ N₂ O₇ : C, 69.95; H, 5.34; N, 4.94.Found: C, 69.74; H, 5.67; N, 4.78.

EXAMPLE 16 Selective removal of failure sequences during non-PASSoligonucleotide synthesis by capping with a diene-modified cappingreagent and subsequent capture of such species on a dienophile resin ormembrane Preparation of 3,5-hexadienoic acid anhydride (74),3,5-hexadienoxyacetic anhydride (75) and trihexadienoxysilyl chloride(76)

Compounds 74, 75, and 76 are prepared by standard methods known in thefield. Compound 74 can be prepared from the 3,5-hexadienol by oxidationto the corresponding hexadienoic acid and subsequent dehydration.Compound 75 is obtained from reaction of iodoacetic anhydride with3,5-hexadienol and compound 76 is a product of the reaction of silicontetrachloride with 3,5-hexadienol. In addition to these methods ofsynthesis, compounds 74, 75, and 76 can be prepared by a variety ofother methods.

Use of compound 75 as capping reagent and subsequent failure removalduring 3'-PEG anchored solution phase synthesis

A solution phase synthesis is performed as described in Example 7 withthe exception that the capping reagent is altered and that a failuresubtraction step is added. During the capping step equal amounts of3,5-hexadienoxyacetic anhydride (75), 2,6-lutidine, andN-methylimidazole are simultaneously injected into the solution andstirred. Maleimide-derivatized polystyrene resin is added to thereaction mixture and stirring is continued. The resin is filtered offand the polymer is precipitated from ether as described in thedetritylation procedure of Example 7.

Use of compound 76 as capping reagent and failure removal duringconventional solid phase synthesis

Conventional solid phase synthesis of DNA, RNA, and modifiedoligonucleotides is carried out according to the specifications given bythe solid phase synthesizer manufacturer with the exception thattri(3,5-hexadienoxy)silyl chloride 76 is substituted for aceticanhydride in the capping reagent. Upon cleavage and deprotection of theoligonucleotide from the support the crude oligonucleotide is taken upin water/acetonitrile and maleimide-derivatized polystyrene is added tothe solution. Upon complete reaction, the resin-bound failure sequencesare filtered off and the product oligonucleotide is further purified ifrequired.

EXAMPLE 17 Use of N-2,7-di(3,5-hexadienoxyacetyl)fmoc protected aminoacid monomers for peptide synthesis by PASS Preparation ofN-2,7-di(3,5-hexadienoxyacetyl)fmoc protected amino acid monomers (86)##STR25## Scheme 19 outlines the synthesis of2,7-di(3,5-hexadienoxyacetyl)fmoc protected amino acids 86. Briefly,Friedel-Crafts acylation of fluorene with chloroacetyl chloride givesthe 2,7-chloroacetylfluorene derivative compound 84. (Leiserson andWeissberger (1955) Org. Synth III, 183). Nucleophilic substitution ofthe dichloride with 3,5-hexadienoxide yields2,7-(3,5-hexadieneoxyacetyl)fluorene, which is converted to the9-methylchloroformate derivative 85 by addition to formaldehyde andsubsequent condensation with phosgene. (Bodansky and Bodansky (1984) inThe Practice of Peptide Synthesis (Springer Verlag, Berlin). Thehexadienoxyacetyl-fmoc chloroformate 85 is then condensed with theN-terminal amino group of a side-chain protected amino acid in standardfashion to yield the 2,7-di(3,5-hexadienoxyacetyl)fmoc protected aminoacid 86. (Bodansky and Bodansky (1984) in The Practice of PeptideSynthesis (Springer Verlag, Berlin). Peptide assembly with2,7-di(3,5-hexadienoxyacetyl)fmoc protected amino acids by PASS

Scheme 20 illustrates peptide assembly by PASS using2,7-di(3,5-hexadienoxyacetyl)fmoc protected amino acids. ##STR26## Thehexadienoxy-fmoc protected amino acid monomer 87 is added to an extendedpeptide chain 88, where W is a peptide protected at the C-terminus withstandard protecting groups, a blocking group, a soluble polymer or adiagnostic detector, each of which can be prepared by standard methods,to give product 89, together with unreacted 88 and excess amino acidmonomer 87. (Bodansky and Bodansky (1984) in The Practice of PeptideSynthesis (Springer Verlag, Berlin). From this mixture product 89 andexcess monomer 87 are captured by Diels Alder cylcloaddition onmaleimide derivatized cellulose. After washing away 88, the product isreleased from the resin by basic reagents typically used to remove thefmoc protecting group. The product 90 is separated from unprotectedreleased excess monomer by extraction and 90 is then ready to undergothe next monomer addition to extend the peptide chain.

EXAMPLE 18 Preparation and use of2,7-di(maleimido)fluorene-9-methylchloroformate for peptide synthesis byPASS ##STR27##

2,7-di(maleimido)fluorene-9-methylchloroformate 91 (Scheme 21) isprepared from the diaminofluorene 92 by condensation with maleicanhydride followed by conversion to the 9-chloroformate derivative 91using standard methods. This protecting group can be used in a PASSpeptide synthesis cycle analogous to Example 17, wherein the resin ishexadiene substituted cellulose, rather than maleimide derivatizedcellulose.

EXAMPLE 19 Peptide assembly using hexadienoxy-Boc protected amino acidsby PASS ##STR28##

The hexadienoxy-Boc protecting group 94 is prepared from 93 by reactionwith 3,5-hexadienol (Scheme 22). Protecting group 94 is then condensedwith the N-terminal amino group of a side-chain protected amino acidusing standard procedures to yield the hexadienoxy-Boc protected aminoacid monomer 95. (Bodansky and Bodansky (1984) in The Practice ofPeptide Synthesis (Springer Verlag, Berlin). Compound 95 can be employedin a PASS synthesis of a peptide analogous to Example 17. As illustratedin Scheme 22, the coupled product 96 is anchored by cycloaddition of thehexadieneoxy-Boc protecting group with maleimide derivatized celluloseand is released using standard acid treatment for Boc removal.

EXAMPLE 20 Peptide Nucleic Acid Preparation by PASS

The PASS synthesis of peptide nucleic acids (PNAs) proceeds analogous tothe cycle described in Example 17, with the difference that thehexadienoxy-fmoc protected monomer is a PNA monomer 98 (Scheme 23).##STR29##

                  TABLE 1                                                         ______________________________________                                        Mobility (R.sub.f) of Alkyl Substituted Tritanols on C18 Reverse Phase.         Solvent    DOT       4-decyloxy-4'-methoxytritanol                                                                DMT                                     ______________________________________                                        acetonitrile                                                                           0         0.52             0.77                                        methanol 0 0.49 0.71                                                          80% acetic acid 0 0.02 0.45                                                 ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        Monomer addition cycle protocol.                                                                     Agent/            Time                                   Step Procedure Solvent Quantity.sup.1 (mL) (min.)                           ______________________________________                                        1.    detritylation                                                                              2.5% DCA   50                                                  in CH.sub.2 Cl.sub.2  9                                                       trihexylsilane 6.4                                                          2. precipitate CH.sub.2 Cl.sub.2 /Et.sub.2 O                                   (twice)                                                                      3. coupling Amidite 4.5 mL (2.0 eq)                                             DCI 1.4 mL (6.0 eq) 25                                                        CH.sub.3 CN 50 mL                                                           4. precipitate Et.sub.2 O                                                     5. oxidation iodobenzene 8 8                                                    diacetate 50                                                                 capping CH.sub.3 CN 6/6/6 5                                                    capping soln..sup.2                                                         6. precipitate (twice) Et.sub.2 O/CH.sub.2 Cl.sub.2                           7. crystallization EtOH 500                                                 ______________________________________                                         .sup.1 Quantities are for 5.0 g of starting PEGnucleoside (loading, 45        μmol/g).                                                                   .sup.2 Capping solution: acetic anhydride, 2,6lutidine, Nmethylimidazole.

                  TABLE 3                                                         ______________________________________                                        Coupling Efficiency (%) for 10 mer of Oligonucleotide                           cycle          ester linker                                                                            amide linker                                       ______________________________________                                        1            99.6      99.2                                                     2 162 123                                                                     3 99.4 98.2                                                                   4 99.5 99.3                                                                   5 99.1 99.2                                                                   6 99.5 99.0                                                                   7 97.1 97.3                                                                   8 98.1 97.8                                                                   9 97.3 97.6                                                                 ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Rates of Cycloaddition of Diene-substituted Tritanols with                      N-Ethylmaleimide.*                                                                  Reaction Conditions    Completion of                                  N-ethyl-     CH.sub.3 CN/H.sub.2                                                                             Reaction (%)                                   Reaction                                                                              maleimide                                                                              O         Time  Rxn 1  Rxn 2                                   #  (eq.) %/% (hours) (30)→(37) (36)→(38)                      ______________________________________                                        1       2        100/0     3     29     20                                         5 36 28                                                                       24 65 57                                                                   2 10 100/0 1 52 34                                                               3 71 51                                                                       5 82 72                                                                    3 10 50/50 1 84 68                                                               3 100  93                                                                     5 N/A 100                                                                ______________________________________                                         *Reactions were carried out at room temperature in deuterated solvents.       The % completion was determined by .sup.1 H NMR analysis of an aliquot        taken directly from the crude reaction mixture. All reactions were carrie     out at a concentration of 0.07 M unless otherwise noted.                 

                  TABLE 5                                                         ______________________________________                                        Rates of Cycloaddition of Thymidine                                             Substituted Tritanols with N-Ethylmaleimide.                                         Time    % Completion                                                 ______________________________________                                        5'-(DHDT)thymidine (31)                                                                1 hour  78                                                             3 hours 100                                                                 5'-(DHDT)thymidine 3'-phosphoramidite (32)                                             1 hour  63                                                             3 hours 96                                                                    5 hours 100                                                                 ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        Recovery of 20k-PEG-dT from an Ultrafiltration                                  Membrane Using an Acetonitrile Solvent System.                                     First Wash    Second Wash                                                                              PEG-dT                                          (μmol PEG-dT) (μmol PEG-dT) Recovered (%)                             ______________________________________                                        Control                                                                              5.41                     98.7%                                           0.25 hour 5.30 0.12 98.9%                                                     1 hour 5.31 0.07 98.2%                                                        4 hour 5.28 0.13 98.7%                                                      ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Recovery of 20k-PEG-dT from an Ultrafiltration                                  Membrane Using Methylene Chloride.                                                 First Wash    Second Wash                                                                              PEG-dT                                          (μmol PEG-dT) (μmol PEG-dT) Recovered (%)                             ______________________________________                                        Control                                                                              5.52                     101.4%                                          0.25 hour 5.19 0.13 97.7%                                                     1 hour 5.32 0.11 99.7%                                                        4 hour 5.33 0.08 99.3%                                                      ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        Recovery of 20k-PEG-dT from a Regenerated                                       Ultrafiltration Membrane Using Acetonitrile.                                        First Wash Soak & Second Wash                                                                           PEG-dT                                        (μmol PEG-dT) (μmol PEG-dT) Recovered (%)                             ______________________________________                                        Control 5.62                      98.7%                                         0.25 hour 4.80 0.90 100.0%                                                    1 hour 4.45 1.20 99.1%                                                        4 hour 4.40 1.20 98.3%                                                        24 hour 4.17 1.40 97.7%                                                     ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Flux Data to Membranes Exposed to Synthesis Solvents.                                               CH.sub.3 CN                                                 rinse of                                                                     CH.sub.3 CN c/c/o/T  CH.sub.3 CN rinse                                       Membrane only exposed DCA/CH.sub.2 Cl.sub.2 of DCA/CH.sub.2 Cl.sub.2        ______________________________________                                        PVDF      0.75    0.83    0.94     0.8                                          polypropylene 7.19 7.45 8.76 8.51                                           ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15  nucl - #eotides                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #The bond between G at position 13       and                                                                                            T at p - #osition 14 is a [3',3'] linkage.                      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - CTAAACGTAA TGGTT              - #                  - #                      - #    15                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10  nucl - #eotides                                               (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #The bond between G at position 8 and                     T at p - #osition 9 is a [3',3'] linkage.                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - CGTAATGGTT                - #                  - #                      - #        10                                                                 __________________________________________________________________________

We claim:
 1. A method for the solution phase synthesis of peptidescomprising: a) reacting an N-terminal protected amino acid monomer unithaving the following formula: ##STR30## wherein R⁵ is selected from H ora protected known amino acid side chain;A is an amino protectinggroup(s); and X is selected from the group consisting of a diene,dienophile, alkyne and disulfide, with a peptide starting material toform a reaction mixture containing a peptide product; and b)partitioning the peptide product from the unreacted peptide startingmaterial, unreacted N-terminal protected amino acid monomer unit,side-products and reagents based on the presence of the N-terminalprotecting group.
 2. The method of claim 1 further comprising:c)repeating steps a) and b) in successive cycles a select number of timesto yield the desired product.
 3. The method of claim 1 wherein A isselected from the group consisting of a urethane, benzyl, acyl andtriphenylmethyl.
 4. The method of claim 3 wherein said urethane is9-fluorenylmethyl carbonyl (Fmoc) or tert-butoxycarbonyl (Boc).
 5. Amethod for the solution phase synthesis of peptides comprising:a)reacting an N-terminal protected amino acid monomer unit with a peptidestarting material to form a reaction mixture containing a peptideproduct; and b) partitioning the pentide product from the unreactedpeptide starting material, unreacted N-terminal protected amino acidmonomer unit, side-products and reagents based on the presence of theN-terminal protecting group, wherein the partitioning is performed byeluting the reaction mixture through a solid support, wherein said solidsupport covalently reacts with said N-terminal protecting group.
 6. Themethod of claim 5 wherein said covalent reaction is a Diels-Alderreaction.
 7. The method of claim 5 wherein said solid support isselected from the group consisting of a resin, membrane and polymer. 8.The method of claim 5 wherein said solid support is selected from thegroup consisting of a hydrophobic reversed phase resin, a thiopropylsepharose resin, a mercurated resin, an agarose adipic acid hydrazideresin, an avidin resin, an ultrafiltration membrane, Tentagel™,polyethylene glycol and an inorganic oxide, selected from the groupconsisting of silica gel, alumina, controlled pore glass and zeolite. 9.The method of claim 8 wherein the hydrophobic reversed phase resin isselected from the group consisting of a C2 to a C18 polystyrene resin.10. A method for the solution phase synthesis of peptides comprising:a)reacting an N-terminal protected amino acid monomer unit with a peptidestarting material to form a reaction mixture containing a peptideproduct; and b) partitioning the peptide product from the unreactedpeptide starting material, unreacted N-terminal protected amino acidmonomer unit, side-products and reagents based on the presence of theN-terminal protecting group, wherein the partitioning is performed byeluting the reaction mixture through a solid support, wherein said solidsupport is derivatized with a group selected from a diene, dienophile,mercaptane and borate.
 11. The method of claim 10 wherein said diene isselected from the group consisting of 3,5-hexadiene.
 12. The method ofclaim 10 wherein said dienophile is maleimide.
 13. The product formed bythe method of claim
 1. 14. The method of claim 1 wherein said diene is3,5-hexadienoxy or sorbic amide.